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354
Differences among mechanisms of ribozyme-catalyzed reactions
Masaki Warashina*†, Yasuomi Takagi†, Wojciech J Stec‡ and
Kazunari Taira*†
The catalytic properties of ribozymes depend on the
sophisticated structures of the respective ribozyme–substrate
complexes. Although it has been suggested that ribozymemediated cleavage of RNA occurs via a rather strictly defined
mechanism, recent findings have clearly demonstrated the
diversity of reaction mechanisms.
Addresses
*Department of Chemistry and Biotechnology, Graduate School of
Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
† National Institute for Advanced Interdisciplinary Research, AIST, MITI,
Tsukuba Science City 305-8562, Japan
‡ Polish Academy of Science, Centre of Molecular and Macromolecular
Studies, Department of Bioorganic Chemistry, Sienkiewicza 112,
90-363 Lodz, Poland
Correspondence: Kazunari Taira; e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:354–362
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
GS
ground state
HDV
hepatitis delta virus
TS
transition state
must be metalloenzymes that require divalent metal ions for
catalysis and that the cleavage mechanism is unique to
ribozymes but common to all types of ribozyme. Recent
advances, however, have revealed examples of metal-ionindependent cleavage by hairpin ribozymes and it has
become clear that multiple mechanisms for cleavage by
ribozymes must exist, depending upon the architecture of
each ribozyme. It has also become clear that the architecture
of HDV ribozyme–substrate complexes can influence the
pKa of the ring nitrogens of cytosine and adenine, with the
resultant perturbed ring-nitrogens being directly involved in
acid/base catalysis [6,7•,8••]. Nevertheless, there is no doubt
that RNA catalysts lacking functional substituents under
physiological conditions exploit metal ions for an appropriate structure and for catalytic function and that the majority
of ribozymes use metal ions as cofactors. The extensive
potential of RNA molecules to act as catalysts and the
sequential events that occur in reactions catalyzed by
ribozymes have been of great interest, and the role of the
catalytic metal ions have been the focus of much experimentation and discussion [9–15,16•,17–19]. Among various
ribozymes studied in the past, we will focus on a few
ribozymes whose enzymatic mechanism became unveiled
over the past year at the atomic level.
Introduction
Catalytic RNAs are called ribozymes and they include
hammerhead, hairpin and hepatitis delta virus (HDV)
ribozymes, group I and II introns, the RNA subunit of
RNase P, and ribosomal RNA [1–5]. Among these
ribozymes, the first two ribozymes to be discovered, by
Altman and Cech, respectively, were the RNA subunit of
RNase P and a group I intron [1–3]. Within the following
five years, small ribozymes, such as hammerhead, hairpin
and HDV ribozymes, were discovered in studies of the
replication, via a rolling-circle mechanism, of certain
viroids, satellite RNAs and an RNA virus [1,4]. These
ribozymes catalyze RNA cleavage/ligation reactions. In the
case of the ribozyme-mediated cleavage of RNA, first a
nucleophile attacks the phosphorus atom at the scissile
phosphate with resultant formation of a pentavalent phosphorus species, which is either a transition state or a
short-lived intermediate, then a leaving oxygen departs
from the phosphorus. The nucleophile can either be an
internal 2′-oxygen in small ribozymes, such as hammerhead, hairpin, and HDV ribozymes, or an external
nucleophile in larger ribozymes, such as the group I intron
and the RNA subunit of RNase P.
Ribozymes are considered to be fossil molecules that originated in a hypothetical prebiotic RNA world. A full
understanding of their mechanisms of action should
enhance our understanding of the evolution of primitive
organisms [1–5]. It has long been assumed that all ribozymes
Reactions catalyzed by the Tetrahymena
ribozyme: a double-metal-ion mechanism
The reaction mechanism of ribozymes that catalyze the
cleavage of RNA phosphodiester linkages, in particular
that of large ribozymes such as group I introns and the catalytic RNA subunit of RNase P, involves external
nucleophiles. By contrast, small ribozymes, such as hammerheads, hairpins and HDV ribozymes, use an internal
nucleophile, namely, the 2′-oxygen of a ribose moiety at
the cleavage site, with the resultant formation of a 3′-terminal 2′-O,3′-O-cyclic phosphate. Despite the large size of
the group I ribozyme of Tetrahymena with its sophisticated
higher-order structure, the extensive efforts made over the
past decade have revealed the details of the mechanism of
the Tetrahymena ribozyme-mediated cleavage of RNAs.
Detailed kinetic and thermodynamic analyses of wild-type
and mutated forms of this ribozyme have helped to define
the reaction mechanism at the atomic level [1,20,21,22••].
Chemical modification at the atomic level has generally
involved introduction of a sulfur atom to replace an oxygen
atom that has the potential to interact with a catalytically
important metal ion. The reduction in the cleavage rate in the
presence of Mg2+ after such a modification (the so-called thio
effect) and the subsequent observation of cleavage with the
phosphorothioate-modified substrate or ribozyme at the same
rate as that of cleavage with the normal RNA components in
the presence of Mn2+ (the so-called manganese rescue effect)
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 1
5′
Exon
G
U (–1) 5′
O
O
3'
Mg 2+
A
Intron
H
–O
pro-Sp
Mg 2+
C
O
2′
G
Ribozyme
2'
O
H
O
3′
P
O
Substrate
A
O
O
3′
O
2′
pro-Rp
O
3′
O
2+
Mg
B
OH
OH
Intron
3′
Current Opinion in Biotechnology
The transition state during the splicing reaction mediated by a
Tetrahymena group I intron. Replacement of a specific oxygen atom
with a sulfur atom followed by metal-dependent cleavage, revealed
three important metal-ion binding sites (A, B and C).
have been taken as evidence that supports the direct coordination of the oxygen atom in question with a metal ion. This
phenomenon can be explained by the hard–soft acid base
(HSAB) rule. According to this rule, a ‘hard acid’, such as
Mg2+, prefers to bind to a ‘hard base’ oxygen atom rather than
to a ‘soft base’ sulfur atom. By contrast, a ‘soft acid’, such as
Cd2+, prefers to bind to a ‘soft base’ sulfur atom. Mn2+ is softer than Mg2+ and, thus, can bind to a soft sulfur atom (as well
as to a hard oxygen atom) and is believed to be the origin of
the manganese rescue effect. Analysis of both the thio effect
and the manganese rescue effect has contributed significantly to our understanding of the mechanism of action of the
Tetrahymena ribozyme. Such analysis has revealed the three
metal-binding sites that are shown in Figure 1 [22••]. Thus, it
is generally accepted that the Tetrahymena ribozyme is a metalloenzyme that operates via the double-metal-ion
mechanism of catalysis, in which Mg2+ at the B site enhances
the deprotonation of the 3′-OH of the guanosine nucleophile
and Mg2+ at the A site stabilizes the leaving 3′-bridging oxygen of U(–1) in the transition state (TS). A third Mg2+ is
postulated to bind at the C site to interact directly with the
2′-OH of the guanosine nucleophile.
Although the above mechanism is, indeed, generally accepted, a linear free-energy analysis of the cleavage of
oligonucleotide substrates with a series of 2′-substituents at
U(–1) suggested that the effect on the catalytic rate of the
355
2′-OH group is larger than might be expected from simple
inductive effects. Specifically, the weaker electron-withdrawing 2′-OH group enhances the chemical cleavage step to
a greater extent than did the stronger electron-withdrawing
2′-F atom of the corresponding 2′-deoxy-2′-fluoro derivative.
Therefore, the possibility of a symmetrical TS, in which the
2′-OH of U(–1) might or might not interact with a metal ion
(as observed at site C), was recently examined [22••].
Despite the absence of lone-pair electrons at the 2′-NH3+
group that might interact with a metal ion, the higher reactivity of the substrate with a 2′-deoxy-2′-NH3+ group than
that of the substrate with a 2′-OH group at U(–1) suggested
that interaction of a metal ion with the 2′-OH of U(–1) might
not be important for catalysis by the Tetrahymena ribozyme.
The higher reactivity of the derivative suggests that donation
of a hydrogen bond from the neighboring 2′-group to the
3′-bridging oxygen might allow specific stabilization of the
TS relative to the ground state (GS), thereby facilitating the
chemical cleavage step. The network of interactions
between the 2′-OH of U(–1), the 2′-OH of A207 and the exocyclic amino group of G22 have been referred to as a catalytic
triad. However, the observation that the chemical cleavage
step with the substrate with a 2′-NH3+ moiety is faster than
that with the 2′-OH group, despite the absence of lone-pair
electrons at the 2′-NH3+ group that can accept a hydrogen
bond from A207, suggests another possibility for the arrangement of active-site groups within this network [22••].
Even though the ribozyme-mediated chemical cleavage
step for the substrate with a 2′-OH group is significantly
(103-fold) faster than that with 2′-H at U(–1), with metalbinding site A being occupied by a Mn2+, the rate constants
for reactions with 3′-deoxy-3′-thio-modified substrates are
similar irrespective of whether there is a 2′-OH group or
2′-H at U(–1). Furthermore, in the presence of Mg2+, with
metal-binding site A unoccupied by a metal ion, the rate
constants for reactions with 3′-deoxy-3′-thio-modified substrates are similar with a 2′-OH group or with 2′-H at U(–1),
indicating that the 2′-OH group at U(–1) does not contribute significantly to the chemical cleavage of the
phosphorus–sulfur bond with a 3′-mercapto leaving group.
Since sulfur is a weaker acceptor of a hydrogen-bond than
oxygen and, furthermore, since sulfur is a significantly better leaving group than oxygen, the 3′-mercapto leaving
group suppresses the catalytic advantage provided by a
hydrogen bond from the 2′-OH in the native TS [22••].
Analysis of the thio effect and rescue experiments have
contributed significantly to elucidation of the mechanism
of action of the large group I ribozyme of Tetrahymena. All
the available data appear to support the refined doublemetal-ion mechanism of catalysis that is shown in Figure 1.
Thio effects and rescue experiments in
reactions catalyzed by hammerhead ribozymes
Metal-ion rescue experiments have also provided considerable information about the sites at which functional
metal ions bind in reactions catalyzed by hammerhead
356
Protein technologies and commercial enzymes
Figure 2
(a)
(b)
Ground state
Transition state
KD
101
[Ca 2+] = 100 mM
100
}
k cat
O
With metal ion
Ca
O
P
O
C17
Ca
Cd
k obs (min -1)
O
H
O (S)
P
P9
O
O
X
[R·M] ‡
R·M
Cd
10-2
C17
O
O
H
O (S)
O
10-1
O
k cleav(PO) = k cleav(PS)
Cd
P9
Native substrate
K GS(PO) = KGS(PS)
K TS(PO) , K TS(PS)
10-3
Phosphorothioate
substrate
10-4
O
Without metal ion
-5
10
(0)
10-8
10-7
10-6
10-5
10-4
10-3
10-2 10-1
[CdCl 2] (M)
C17
O
k (PO) = k (PS)
O
O
O
H
O P O (S)
O
R
C17
O
O
H
O (S)
P
O
R‡
Current Opinion in Biotechnology
Evidence for the identical affinity of a Cd2+ for the native substrate and a
phosphorothioate substrate. (a) Titration with Cd2+ of the ribozymecatalyzed cleavage of each substrate (native substrate, solid line;
phosphorothioate substrate, dashed line) with 100 mM Ca2+ as
background (50 mM MES-Na [pH 6], 37°C). The black arrows indicate
the apparent KD for each ribozyme–substrate complex (left).
(b) Thermodynamic cycles for the rescue of cleavage of the native and/or
the phosphorothioate substrate by addition of Cd2+. Binding of a Cd2+ to
the Rp position of the scissile phosphate in the scheme proposed by
other groups [24,25•] is unlikely. ‘Without metal ion’ means that the
cleavage site is empty but other metal-binding sites are occupied by
background metal ions (right). KGS and KTS are actual KD in the ground
state and the transition state, respectively. KGS(PO) = KGS(PS) can be
estimated from the apparent KD values. The relation of k(PO) = k(PS) was
demonstrated even in an inactivated hammerhead-ribozyme–substrate
complex [47]. R, ribozyme–substrate complex; M, metal ion.
ribozymes [9,23,24,25•,26••]. In such experiments, analogs
of hammerhead ribozymes with phosphorothioate modifications were synthesized, with a sulfur atom replacing
either a bridging or a non-bridging oxygen at the internucleotide phosphorus atom at a preselected position. In
some cases, such substitution resulted in the loss of activity in the presence of Mg2+, which is generally accepted as
the natural metal cofactor. Addition of even trace amounts
of either Mn2+ or Cd2+, however, restored catalysis. These
results supported the hypothesis that the cleavage process
requires direct coordination of a Mg2+ with the phosphate
oxygen and in the case of phosphorothioate analogs
involvement of interaction of a thiophilic metal ion with
the sulfur atom. Specifically, in the presence of Mg2+ or
Ca2+ ions, the rate of the ribozyme-catalyzed cleavage of a
phosphorothioate substrate, in which one of the non-bridging oxygens (the pro-Rp oxygen atom) had been replaced
by sulfur, was nearly four orders of magnitude lower than
that of the reaction with the natural substrate (thio effect).
However, the rates of the ribozyme-catalyzed reactions
with the natural substrate and the phosphorothioate substrate were enhanced by Cd2+ (Cd2+ rescue). Such results
have generally been considered to provide evidence of the
direct interaction of the sulfur atom at the Rp position of
the cleavage site with the added Cd2+; however, that conclusion has been controversial. Results from our laboratory
have indicated that the added Cd2+ binds at the P9 site and
that, contrary to the conclusion reached by other investigators, Cd2+ does not interact with the sulfur atom at the Rp
position of the scissile phosphate in the GS or in the
TS [26••]. Our intepretation is based on the analysis shown
in Figure 2, as discussed below.
We investigated the effects of Cd2+ against a background
of Ca2+. In the case of cleavage of unmodified and phosphorothioate-modified substrates, each curve of the
logarithm of activity versus the logarithm of the concentration of Cd2+ can be divided into three regions. In the
absence of Cd2+, the ribozyme–substrate complex
remains in a less active state in the presence of background Ca2+. Upon addition of Cd2+, Cd2+ replaces
pre-existing Ca2+ within the ribozyme–substrate complex
and, in proportion to the extent of such replacement, the
activity of the ribozyme increases linearly until the relevant Ca2+ has been completely replaced by Cd2+. After
the completion of the replacement of Ca2+ by Cd2+(at
Cd2+ concentrations greater than the apparent KD of
~3 mM; Figure 2, left), the ribozyme–substrate complex
enters the activated state with bound Cd2+.
From the titration curves in Figure 2, an estimate can be
made of respective kinetic and thermodynamic parameters
in the thermodynamic cycle, as follows. As the pre-existing
Ca2+ within the ribozyme–substrate complex was replaced
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
357
by Cd2+ with an identical apparent KD for both normal (PO)
and phosphorothioate (PS) substrates, the binding affinity of
the added Cd2+ to each respective complex in the GS should
be the same: KGS(PO) = KGS(PS). The rate of the cleavage in
the activated complex [R•M]‡ is 1 min–1 at saturating Cd2+
and is the same for both the natural and phosphorothioate
substrates: kcleav(PO) = kcleav(PS). Furthermore, the rate of
nonenzymatic cleavage of the P–O bond in the two substrates is similar: k(PO) = k(PS).
hydroxide that acts as a general base catalyst. Discovery of
this phenomenon provided the first direct demonstration
that a nucleoside can act as an acid–base catalyst in RNA.
As a result, as shown in Figure 3a, the curve showing the
dependence on pH of the self-cleavage of the precursor
HDV genomic ribozyme has a slope of unity at pH values
below 7 (the activity increases linearly as pH increases
with a slope of +1) and then, at higher pH values, the
observed rate constant is insensitive to pH.
The application of thermodynamics and TS theory permits
the calculation of KTS(PO) and KTS(PS). Because three out
of four parameters were the same for the natural substrate
and the phosphorothioate substrate — KGS(PO) = KGS(PS),
kcleav(PO) = kcleav(PS) and k(PO) = k(PS) — the remaining parameter should also be the same for the two substrates:
KTS(PO) = KTS(PS). It is apparent, therefore, that the affinity of Cd2+ for the ribozyme–substrate complex is the same
for both complexes not only in the GS, KGS(PO) = KGS(PS),
but also in the TS, KTS(PO) = KTS(PS), irrespective of
whether the cleavage site includes a regular phosphate or a
modified phosphorothioate moiety.
The slope of unity at low pH is consistent with an increase,
with pH, in the concentration of the metal hydroxide,
which acts as the general base upon deprotonation
(Figure 3b), and a constant amount of the functional protonated form of C75, which acts as the general acid
(Figure 3c). The slope of zero from pH 7 to pH 9 indicates
that the concentration of the metal hydroxide [B:] increases, whereas the concentration of the protonated C75 [AH]
decreases by the same amount [8••]. In agreement with
this interpretation, when C75 was replaced by uracil, the
resultant C75→U mutant, which was unable to assist in the
transfer of a proton, did indeed lack catalytic activity. The
activity of the C75→U mutant could be restored, however,
by the addition of imidazole whose protonated form (imidazolium ion) is known to act as a good proton donor
[7•,8••]. Another mutant, C75→A, in which the ring nitrogen N1 at A75 has a slightly lower pKa than the
corresponding ring nitrogen N3 of C75, supported selfcleavage activity, albeit with reduced efficiency. The
observed pKa of the C75→A mutant was slightly lower
than that of the wild-type C75 ribozyme, supporting the
interpretation that a nucleic acid base acts as a catalyst in
HDV ribozyme-catalyzed reactions as shown in Figure 3.
We must conclude that Cd2+ does not interact directly with
the sulfur atom at the Rp position of the scissile phosphate
either but binds to the P9/G10.1 motif to restore the catalytic activity of the cleavage site [25•,26••]. Consequently,
observations of the thio effect and manganese rescue effect
themselves may not provide decisive arguments in support
of the hypothesis that direct coordination of a metal ion
with the pro-Rp oxygen at the cleavage site is a precondition for hammerhead ribozyme-catalyzed reactions [9,26••].
Even a single substitution of sulfur for oxygen has serious
steric effects, rather than the expected kinetic thio effect,
in reactions that are catalyzed by RNase P [27•]. With a
3′-deoxy-3′-S-phosphorothiolate-modified precursor tRNA,
which can be processed by RNase P, the cleavage site shifts
from the expected phosphorothiolate-modified site to the
adjacent unmodified phosphodiester linkage in the 5′
direction. The bulkiness of the sulfur atom might prevent
the correct positioning of the scissile bond at the active site.
Hepatitus delta virus ribozyme-catalyzed
reactions
Recent reports by three groups have revolutionized our
understanding of the mechanism of HDV ribozyme-catalyzed reactions [6,7•,8••]. For the cleavage of
phosphodiester bonds, the nucleophile must be deprotonated and the leaving group must be protonated or
stabilized by a metal ion. As shown in Figure 1, the developing negative charges in the TS in the Tetrahymena
ribozyme-catalyzed reaction are stabilized by direct interactions with metal ions. The novel finding with respect to
the mechanism of catalysis by the HDV ribozyme is that
the pKa of C75 is perturbed to neutrality in the
ribozyme–substrate complex and, more importantly, C75
acts as a general acid catalyst in combination with a metal
Further convincing evidence for this model comes from
the observation that, in the absence of divalent metal ions
(in the absence of base B; Figure 3b) and in the presence
of a high concentration of monovalent cations (~2 M NaCl
and 1 mM EDTA), the logarithm of the observed rate constant decreased with pH with a slope of –1 (as shown in
Figure 3c) with only general acid catalysis operative (in the
absence of the metal hydroxide as a general base). This
type of profile clearly demonstrates that the observed pKa
for self-cleavage results from the general acid rather than
from the general base. It is noteworthy that [Co(NH3)6]3+
which is known to be a substitutionally inert transition
metal complex (kex < 10–10 s–1 even at acidic pH) inhibited the Mg2+-catalyzed reaction in a competitive fashion,
suggesting that [Co(NH3)6]3+ might bind to the same site
as the functional Mg2+ with outer-sphere coordination but
does not ionize. The similar rate constants in the presence
of Ca2+ and of Mg2+ are also consistent with the action of a
hydrated metal ion as a Brönsted base rather than a Lewis
acid in the HDV ribozyme.
There was a significant, apparent D2O solvent isotope
effect over the entire range of pH values, confirming that
transfer of a proton occurs in the TS (the ratio of rate
358
Protein technologies and commercial enzymes
Figure 3
pKa = 6
pKa = 11
(d)
5'
log k
5'
O
C21
O
O
O
O
O
P O
H
H N
O
C22
H+
0.7
O H OH
P O
Mg
H
O
G-1
H
N
C75 O
N
3'
U-1
a
=
1
O H
Mg 2+
H
NH2
pKa = 11
H
:
H
O
H
O
Mg 2+
0.6
3'
(b)
:
O
O
pH
log [B:]
pK
1.4
pK a
(a)
a
pK
0.5
H
=
6.1
N
Mg 2+
N
O
C75
NH2
O
N
N
H
O
C75
Mg 2+
0.4
0
5
10
15
pK a
(e)
pH
3.5-fold
pKa = 6
NH2
log [AH]
6.5-fold
log k
(c)
8.5-fold
N
N
C75
H
O
NH2
N
O
N
H2O
D2O
C75
5
pH
10
pL
Current Opinion in Biotechnology
Kinetic analysis of HDV ribozyme-catalyzed reactions. (a) A schematic
replotting of the pH-rate profile of HDV ribozyme-catalyzed reactions
[8••]. This profile has pKa values of 6 and 11. The solid line and the
dotted line represent the experimentally obtained curve and the
theoretical curve, respectively. This profile can be explained by a
combination of the dependence on pH of the concentration of the active
base [B:], as shown in (b), and of the concentration of the active acid
[AH], as shown in (c). (b) The logarithm of the concentration of a general
base catalyst in an active form is plotted against pH. The pKa is 11. (c)
The logarithm of the concentration of a general acid catalyst in an active
form is plotted against pH. The pKa is 6. (d) Isotope effects on the
acidities of phenols and alcohols as a function of their acid strength, pKa
(for details, see [9,28,29]). (e) Theoretical curves for the activity of the
HDV ribozyme in H2O and D2O. The pKa values in H2O and in D2O for
C75 of 6.1 and 6.5, respectively, were taken from the experimental data
[8••]. Using the pKa in H2O for a magnesium-bound water molecule as
being 11.4, we calculated the corresponding pKa values in D2O, from
the linear plot in (d), to be 12 [9]. Theoretical curves were plotted on the
assumption that the value of the intrinsic isotope effect was 2. The
following equation was used to plot the graph of pL versus log(rate):
log kobs = log kmax – log (1 + 10[pKa(base) – pL]) – log (1 + 10[pL – pKa(acid)]) –
log α. Where kmax is the rate constant in the case of all acid and base
catalysts in active forms, and α is the coefficient of the intrinsic isotope
effect, that is, kmax(H O)/kmax(L O). In the pH profile, α = 1; in the pD profile,
2
2
α = 2 under the above mentioned assumption.
constants, kcat(H O):kcat(D O), being as high as 10; see
2
2
[8••]). Moreover, because, firstly, the observed pKa for
self-cleavage is that for the general acid and secondly,
the overall rate-limiting step appears to be the cleavage
of the bond between the phosphorus and the 5′-leaving
oxygen (see below and Figure 4), the transferred proton
in the TS must be that of C75. As the concentration of
the protonated C75 in H2O can be higher than that in
D2O at a fixed pH, the difference in pKa (∆pKa), which
equals log(KaH2O/KaD2O), must be taken into account
when the D2O solvent isotope effect is calculated
[9,28,29]. It is possible to estimate the relative concentrations of the active species in H2O and in D2O because
a linear relationship exists between them, as shown in
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 3d [9]. Under the conditions of measurements,
the pKa of C75 is 6.1 and, thus, the estimated ∆pKa turns
out to be 0.53, as indicated by the dashed arrow in
Figure 3d. Indeed, in the pD-logk profile, the pKa is
shifted upward by ~0.4 ± 0.1 pH units, consistent with
the estimated value and with proton transfer by a ring
nitrogen [8••]. If we take the pKa of C75 as 6.1 in H2O
and 6.5 in D2O, and the pKa values for magnesiumbound water to be 11.4 and 12.0, respectively, and if we
assume that the value of the intrinsic D2O solvent isotope effect is two, we can generate theoretical curves for
HDV-catalyzed reactions in H2O and D2O, as shown in
Figure 3e. The agreement of our theoretical curves with
the observed curves [8••] confirms that proton transfer
does indeed occur from protonated C75 in the P–(5′–O)
bond-cleaving TS. This conclusion is different from the
case of hammerhead ribozymes because, in that case, the
observed isotope effect (k cat(H O)/k cat(D O) = 4.4; see
2
2
[9,28,29]) can be fully explained by the difference in relative concentrations of the active species in H2O and in
D2O, with an intrinsic D2O solvent isotope effect being
zero [9,28,29].
Thus, in the case of HDV ribozyme-catalyzed reactions, all
the available data support the reaction mechanism shown
in Figure 3a. The donation of a proton is favored by this
model, which involves an acid with an optimal pKa of 7.
The ability of the HDV ribozyme to perform general
acid–base catalysis effectively under physiological conditions appears to be unique among known ribozymes.
Importance of acid catalysis in reactions
catalyzed by hammerhead and HDV ribozymes
Nonenzymatic hydrolysis, as well as small-ribozyme-mediated hydrolysis, of RNAs occurs via nucleophilic attack by
the 2′-oxygen that generates the pentacoordinate intermediate or TS (via the first putative TS [TS1]). Such attack is
followed by the cleavage of the bond between the phosphorus and the 5′-oxygen leaving group (via the second
putative TS [TS2]; Figure 4). Among the two putative
TSs, TS2 is the overall rate-limiting TS (attack by the
2′-OH on the phosphorus atom is easier than cleavage of
the P–O[5′] bond and, thus, TS2 always has higher energy
than TS1). This conclusion was confirmed in experiments
with an RNA analog with a 5′-mercapto leaving group. If
the formation of the intermediate were the rate-limiting
step (i.e. if TS1 were to be a higher-energy state than
TS2), the phosphorothiolate substrate should be
hydrolyzed at a rate similar to the rate of the hydrolysis of
the natural RNA, because the 5′-bridging phosphorothiolate linkage would not be expected to enhance the attack
by the 2′-oxygen. In contrast, if the decomposition of the
intermediate were the rate-limiting step (i.e. if TS2 were a
higher-energy state than TS1), we would expect that the
phosphorothiolate substrate would be hydrolyzed much
more rapidly than the natural RNA because the pKa of a
thiol is more than 5 units lower than that of the corresponding alcohol. Several groups have confirmed that the
359
phosphorothiolate-modified substrate is significantly more
reactive than the natural RNA in nonenzymatic hydrolytic
reactions [9,10] and, thus, TS2 is indeed always a higherenergy state than TS1 in the cleavage of natural RNAs.
In the case of the hammerhead-ribozyme-catalyzed reactions, the RNA substrate with a 5′-mercapto leaving group
is extremely reactive and handling is difficult [30,31]. In our
laboratory, however, we recently succeeded in measuring
rate constants of 5′-phosphorothiolate-modified and natural
substrates under controlled conditions (measurements
were performed under conditions such that curves of pH
versus log[rate] had a slope of unity for both substrates, and
all undesired side reactions could also be avoided; Y Takagi
and K Taira, unpublished data). As the 5′-phosphorothiolate analog of the substrate was hydrolyzed by hammerhead
ribozymes at significantly higher rates than those measured
with the natural RNA substrate, in agreement with our previous conclusion [31], TS2 must also be a higher-energy
state than TS1 in these ribozyme-catalyzed reactions.
Because the overall transition-state structure in the hydrolysis of RNA is TS2, regardless of whether the reaction
proceeds via a concerted one-step mechanism or via a twostep mechanism with a stable pentacoordinate
intermediate, it is important that any enzyme that catalyzes
the hydrolysis of RNA should stabilize TS2 by donating a
proton to the 5′-leaving oxygen or by ensuring coordination
of a metal ion to the leaving oxygen.
The above scenario should also be applicable to HDVribozyme-catalyzed reactions and, thus, the removal of the
possibility of acid catalysis in the C75→U mutant suppressed the enzymatic activity. By contrast, self-cleavage
of the HDV ribozyme in the absence of divalent cations,
but in the presence of high concentrations of monovalent
cations, suggests that, at high concentrations, monovalent
cations can replace divalent cations in the tertiary folding
of the RNA. Thus, divalent cations are not absolutely
essential for the folding or cleavage activity of the HDV
ribozyme, although a functional Mg2+ does participate in
the cleavage process under physiological conditions.
Metal ion involvement in hammerhead
ribozyme-catalyzed reactions
Base catalysis mediated by Mg2+-hydroxide was postulated as
the mechanism of the chemical cleavage step in reactions catalyzed by a hammerhead ribozyme on the basis of the original
profiles of pH versus the rates of various metal ion-catalyzed
reactions of hammerhead ribozymes [32]. The single-metalion mechanism, in which Mg2+-hydroxide acts as a general
base and abstracts a proton from the 2′-OH internal nucleophile [32], is supported by the finding that no metal ion was
found close to the 5′-leaving oxygen in the crystallographic
structure of a freeze-trapped conformational intermediate of a
hammerhead ribozyme [33,34]. In the more recently determined crystal structure of a modified hammerhead ribozyme
[35], however, Co2+ was located close to the 5′ oxygen atom of
the scissile phosphate. Based upon the crystallographic
360
Protein technologies and commercial enzymes
Figure 4
5′
5′
C21
TS2
O
O
O P
O H
H
N
O
H
N
C22
C75
5′
O
O P
O P
O
H
O H
N
O
H
N
C22
Relative energy
C75
N
3′
O
O
P
O
O
R
H
O
Mg
H
2
G1
O H
3′
O
TS2
G1
5′
O H
O
OH
O−
O
Mg
O
G1
P
O
H N H
TS1
O H
C75
N
R
P O
H
H N
O
H
N
C22
C75 O
O
P
O
N
+
O
3′
O H
H
O
H
Mg
2
HO
O
O
O
O
+
O
U-1
O
3′
k1
U-1
O
O
O
O
5′
3′
O H OH
O
Mg
O
R
C21
k2
O
O
O
TS1
O
5′
U-1
O
C21
N
3′
5′
U-1
O
G1
O H
3′
k1
P(V)
k2
k-1
N
R
Current Opinion in Biotechnology
Energy diagram for cleavage by a ribozyme (HDV ribozyme) of its substrate. The rate-limiting step of the reaction with the normal substrate is the
cleavage of the P-(5′-O) bond. The transition state structures of TS1 and TS2 are also shown.
structure, molecular dynamic simulations led to the proposal
of a new and alternative single-metal-ion mechanism. In the
proposed mechanism, one Mg2+ coordinates directly and
simultaneously to the pro-Rp oxygen and the adjacent oxygen
of the attacking hydroxyl group at the cleavage site, acting as
a Lewis acid to enhance the attack by the 2′-OH moiety on
the phosphorus atom. In addition, it was also proposed that
one of the outer-sphere water molecules that surround the
metal ion is located such that it might act as a general acid to
donate a proton to the 5′-leaving group [36]. As mentioned
above, however, three independent groups recently found no
evidence for the direct coordination of a metal ion to the proRp oxygen, at least in the GS [25•,26••,37]. It was also noted
that the absence of such an interaction could explain the
absence of a thio effect in the hammerhead-catalyzed cleavage of a phosphorodithioate linkage [37]. Our analysis,
depicted in Figure 2, also did not support such an interaction
in the TS [26••].
In contradiction, several reports have now appeared that provide experimental evidence for a double-metal-ion
mechanism of catalysis in hammerhead-catalyzed reactions.
Studies on solvent isotope effects and kinetic analysis of a
modified substrate with a 5′-mercapto leaving group at the
cleavage site have lent strong support to the double-metalion mechanism of catalysis [28,31]. Furthermore,
hammerhead-ribozyme-mediated cleavage has been analyzed as a function of the concentration of La3+ ions in the
presence of a fixed concentration of Mg2+ ions. In this way,
the role of lanthanum could be monitored and the metal ions
could be identified that were directly involved in the cleavage reaction [38,39]. The resultant bell-shaped curve that
originated from activation by the 5′-oxygen-bound La3+ and
deactivation by the 2′-oxygen-bound La3+, shown in
Figure 5, was used to support the proposed double-metal-ion
mechanism of catalysis. Other studies, however, demonstrated that binding of a metal ion (the P9 metal ion) to the
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 5
C
O
Activity of ribozyme
O
O
O
P
Mg 2+ O
O
O–
O
C
O
Mg2+
O
C
O
O
3+
O
3+
La
O
P
O
O
P
Mg 2+
La
O
3+
La
O–
O
O–
O
LaCl3 (µM)
Current Opinion in Biotechnology
A bell-shaped titration curve for cleavage by hammerhead
ribozymes. Both the wild-type ribozyme and the ribozyme with a
7-deaza group at G10.1 generated the same bell-shaped titration
curve (for details, see [38,39,40•]).
pro-Rp oxygen (P9 oxygen) of the phosphate moiety of
nucleotide A9 and to N7 of nucleotide G10.1 is critical for
efficient catalysis, despite the considerable distance (~20 Å)
between the P9 metal ion and the phosphorus of the scissile
phosphodiester group in the GS. In fact, it was demonstrated that an added Cd2+ binds first to the pro-Rp phosphoryl
P9 oxygen and not to the pro-Rp phosphoryl oxygen at the
cleavage site [25•,26••]. Therefore, it was difficult to exclude
completely the possibility that La3+ ions might have replaced
the P9 metal ion and, as a result, might have created conditions represented by the bell-shaped curve. In order to clarify
this situation, we examined a modified hammerhead
ribozyme that included an N7-deazaguanine residue instead
of G10.1. Because the data from our experiments with the 7deaza-ribozyme were free from potential artifacts that might
have resulted from the binding of a La3+ to N7 at G10.1, the
bell-shaped curve obtained for 7-deaza-ribozyme upon the
addition of La3+ to a reaction mixture that contained Mg2+,
as reproduced in Figure 4, strongly supports the proposal that
a double-metal-ion mechanism operates in the cleavage reactions catalyzed by hammerhead ribozymes [40•]. Recent
ab initio molecular orbital calculations also support the double-metal-ion mechanism [41].
Conclusions: diversity among the mechanisms
of ribozyme-catalyzed reactions
It has been well established that the Tetrahymena ribozyme is
a metalloenzyme that operates according to the doublemetal-ion mechanism of catalysis. In general, as indicated
above, other ribozymes also require metal ions for catalysis.
Metal ions are almost certainly needed to induce the folding
of each ribozyme into its active conformation, and the folded
ribozyme might recruit metal ions at correct positions for
direct participation in the chemical cleavage process, as in the
case of the Tetrahymena ribozyme. Alternatively, the folded
RNA structure might itself play a role in the cleavage
361
mechanism [16•,37], as in the case of the HDV ribozyme. In
this latter ribozyme, the folded RNA perturbs the pKa of C75
and, as a result, C75 can perform effective general-acid catalysis under physiological conditions. Evidence that the folded
RNA structure itself functions in the cleavage mechanism
comes from studies of hairpin ribozymes. Three independent
groups have demonstrated that the hairpin ribozyme is almost
fully functional when [Co(NH3)6]3+ replaces Mg2+ [42–44].
The studies of cleavage induced by the exchange-inert
[Co(NH3)6]3+ complex have been extended to include nonmetallic compounds, and hairpin ribozyme-mediated
cleavage has been induced both by aminoglycoside antibiotics and by polyamines [45]. The mechanism of catalysis of
the simplest and most extensively studied hammerhead
ribozyme remains controversial. The activities of hammerhead ribozymes under physiological conditions apparently
depend on Mg2+. Nevertheless, high concentrations of monovalent metal ions (1–4 M monovalent cations, such as Li+,
Na+, and NH4+) can perform the functions of divalent metal
ions [46]. Furthermore, the cleavage activity of hammerhead
ribozymes in the absence of divalent cations suggests that
divalent cations are not absolutely essential for folding or
cleavage in hammerhead ribozyme-catalyzed reactions, as is
also the case for the HDV ribozyme. It remains to be determined whether such high concentrations of monovalent
metal ions might stabilize the substantial negative charge that
develops on the 5′-oxygen atom of the leaving group in hammerhead ribozyme-catalyzed reactions.
In conclusion, the evidence that has accumulated over the
past two years clearly demonstrates the diversity of
ribozyme-catalyzed reactions. Nevertheless, acid-base
catalysis appears to operate in every case even if many
details remain to be clarified.
Acknowledgement
M Warashina and Y Takagi contributed equally to the work. WJ Stec thanks
MITI for the financial support that paid for his three-month stay at the
Laboratory of Molecular Biology of the National Institute of Advanced
Interdisciplinary Research in Tsukuba.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1999.
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Cech TR, Zaug AJ, Grabowski PJ: In vitro splicing of the ribosomal RNA
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the excision of the intervening sequence. Cell 1981, 27:487-496.
3.
Guerrier-Takada C, Gardiner K, Marsh T, Pace N, Altman S: The RNA
moiety of ribonuclease P is the catalytic subunit of the enzyme.
Cell 1983, 35:849-857.
4.
Symons RH: Small catalytic RNAs. Annu Rev Biochem 1992,
61:641-671.
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Yarus M: Boundaries for an RNA world. Curr Opin Chem Biol 1999,
3:260-267.
6.
Ferré-D’Amaré AR, Zhou K, Doudna J: Crystal structure of a
hepatitis delta virus ribozyme. Nature 1998, 395:567-574.
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Protein technologies and commercial enzymes
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Perrotta AT, Shih I, Been MD: Imidazole rescue of a cytosine
•
mutation in a self-cleaving ribozyme. Science 1999, 286:123-126.
A chemical group within HDV RNA is shown, for the first time, to be able to
act as a true catalyst in a ribozyme reaction.
8.
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Nakano S, Chadalavada DM, Bevilacqua PC: General acid-base
catalysis in the mechanism of a hepatitis delta virus ribozyme.
Science 2000, 287:1493-1497.
This paper provides a complete analysis of HDV ribozyme-catalyzed reactions. The most important finding is that C75 acts as a general acid catalyst
rather than as a general base catalyst. The importance of the acid catalyst
for stabilization of the leaving group is also emphasized.
9.
Zhou DM, Taira K: The hydrolysis of RNA: from theoretical
calculations to the hammerhead ribozyme-mediated cleavage of
RNA. Chem Rev 1998, 98:991-1026.
10. Kuimelis RG, McLaughlin LW: Mechanism of ribozyme-mediated
cleavage. Chem Rev 1998, 98:1027-1044.
11. Stage-Zimmermann TK, Uhlenbeck OC: Hammerhead ribozyme
kinetics. RNA 1998, 4:875-889.
12. Doudna JA: Ribozymes: the hammerhead swings into action. Curr
Biol 1998, 8:R495-R497.
13. Walter NG, Burke JM: The hairpin ribozyme: structure, assembly
and catalysis. Curr Opin Chem Biol 1998, 2:24-30.
14. Carola C, Eckstein F: Nucleic acid enzymes. Curr Opin Chem Biol
1999, 3:274-283.
28. Sawata S, Komiyama M, Taira K: Kinetic evidence based on solvent
isotope effects for the nonexistence of a proton-transfer process
in reactions catalyzed by a hammerhead ribozyme: implication to
the double-metal-ion mechanism of catalysis. J Am Chem Soc
1995, 117:2357-2358.
29. Kumar PKR, Zhou DM, Yoshinari K, Taira K: Mechanistic studies on
hammerhead ribozymes. In Catalytic RNA, Nucleic Acids and
Molecular Biology, vol 10. Edited by Eckstein F, Lilley DMJ. Berlin:
Springer-Verlag; 1996:217-230.
30. Zhou DM, Usman N, Wincott FE, Matulic-Adamic J, Orita M,
Zhang LH, Komiyama M, Kumar PKR, Taira K: Evidence for the ratelimiting departure of the 5′′-oxygen in nonenzymatic and
hammerhead ribozyme-catalyzed reactions. J Am Chem Soc
1996, 118:5862-5866.
31. Zhou DM, Zhang LH, Taira K: Explanation by the double-metal-ion
mechanism of catalysis for the differential metal ion effects on the
cleavage rates of 5′′-oxy and 5′′-thio substrates by a hammerhead
ribozyme. Proc Natl Acad Sci USA 1997, 94:14343-14348.
32. Dahm SC, Derrick WB, Uhlenbeck OC: Evidence for the role of
solvated metal hydroxide in the hammerhead cleavage
mechanism. Biochemistry 1993, 32:13040-13045.
33. Scott WG, Murray JB, Arnold JRP, Stoddard BL, Klug A: Capturing
the structure of a catalytic RNA intermediate: the hammerhead
ribozyme. Science 1996, 274:2065-2069.
15. Li Y, Breaker RB: Deoxyribozymes: new players in the ancient
game of biocatalysis. Curr Opin Struct Biol 1999, 9:315-323.
34. Wedekind JE, McKay DB: Crystallographic structures of the
hammerhead ribozyme: relationship to ribozyme folding and
catalysis. Annu Rev Biophys Biomol Struct 1998, 27:475-502.
16. Lilley DMJ: Structure, folding and catalysis of the small nucleolytic
•
ribozymes. Curr Opin Struct Biol 1999, 9:330-338.
The importance of RNA folding in ribozyme-mediated reactions is summarized.
35. Murray JB, Terwey DP, Karpeisky A, Usman N, Beigelman L, Scott
WG: The structural basis of hammerhead ribozyme self-cleavage.
Cell 1998, 92:665-673.
17.
36. Torres RA, Bruice TC: The mechanism of phosphodiester
hydrolysis: near in-line attack conformations in the hammerhead
ribozyme. J Am Chem Soc 2000, 122:781-791.
Scott WG: RNA structure, metal ions, and catalysis. Curr Opin
Chem Biol 1999, 3:703-709.
18. Strobel SA: A chemogenetic approach to RNA function/structure
analysis. Curr Opin Struct Biol 1999, 9:346-352.
37.
19. Westhof E: Chemical diversity in RNA cleavage. Science 1999,
286:61-62.
Murray JB, Scott WG: Does a single metal ion bridge the A-9 and
scissile phosphate groups in the catalytically active hammerhead
ribozyme structure? J Mol Biol 2000, 296:33-41.
20. Weinstein LB, Jones BC, Cosstick R, Cech TR: A second catalytic
metal ion in group I ribozyme. Nature 1997, 388:805-808.
38. Pontius BW, Lott WB, von Hippel PH: Observations on catalysis by
hammerhead ribozymes are consistent with a two-divalent-metalion mechanism. Proc Natl Acad Sci USA 1997, 94:2290-2294.
21. Shan S, Yoshida A, Sun S, Piccirilli JA, Herschlag D: Three metal
ions at the active site of the Tetrahymena group I ribozyme. Proc
Natl Acad Sci USA 1999, 96:12299-12304.
39. Lott WB, Pontius BW, von Hippel PH: A two-metal-ion mechanism
operates in the hammerhead ribozyme-mediated cleavage of an
RNA substrate. Proc Natl Acad Sci USA 1998, 95:542-547.
22. Yoshida A, Shan S, Herschlag A, Piccirilli A: The role of the cleavage
•• site 2′′-hydroxyl in the Tetrahymena group I ribozyme reaction.
Chem Biol 2000, 7:85-96.
Replacement of the leaving oxygen and the 2′-OH by a sulfur atom and an
amino group, respectively, and detailed kinetic analysis are used to analyze
a role of the 2′-OH group in catalysis.
40. Nakamatsu Y, Warashina M, Tanaka Y, Yoshinari K, Taira K: Significant
•
activity of a modified ribozyme with N7-deazaguanine at G10.1:
implication to the double-metal-ion mechanismof catalysis in
reactions catalyzed by hammerhead ribozymes. Genes Cells
2000, 5:in press.
A detailed bell-shaped curve for the activation of cleavage by the added La3+
ions is used to explain the double-metal-ion mechanism.
23. Warashina M, Zhou DM, Kuwabara T, Taira K: Ribozyme structure
and function. In Comprehensive Natural Products Chemistry, vol 6.
Edited by Söll D, Nishimura S, Moore PB. Oxford: Elsevier Science
Ltd.; 1999:235-268.
24. Scott EC, Uhlenbeck OC: A re-investigation of the thio effect at the
hammerhead cleavage site. Nucleic Acids Res 1999, 27:479-484.
25. Wang S, Karbstein K, Peracchi A, Beigelman L, Herschlag D:
•
Identification of the hammerhead ribozyme metal ion-binding site
responsible for rescue of the deleterious effect of a cleavage site
phosphorothioate. Biochemistry 1999, 38:14363-14378.
This paper provides evidence that a metal ion does not bind to the pro-Rp
oxygen of the scissile phosphate bond in the ground state.
26. Yoshinari K, Taira K: A further investigation and reappraisal of the
•• thio effect in the cleavage reaction catalyzed by a hammerhead
ribozyme. Nucleic Acids Res 2000, 28:1730-1742.
Evidence is provided that, contrary to the conclusion reached by other investigators, a metal ion does not interact with the sulfur atom at the Rp position
of the scissile phosphate either in the ground state or in the transition state.
27.
•
Warnecke JM, Sontheimer EJ, Piccirilli JA, Hartmann RK: Active site
constraints in the hydrolysis reaction catalyzed by bacterial
RNase P: analysis of precursor tRNAs with a single 3′′-Sphosphorothiolate internucleotide linkage. Nucleic Acids Res
2000, 28:720-727.
A good example of the large side effects of phosphorothiolate substitution
at the cleavage site.
41. Lyne PD, Karplus M: Determination of the pKa of the 2′′-hydroxyl
group of a phosphorylated ribose: implications for the mechanism
of hammerhead ribozyme catalysis. J Am Chem Soc 2000,
122:166-167.
42. Hampel A, Cowan JA: A unique mechanism for RNA catalysis: the
role of metal cofactors in hairpin ribozyme cleavage. Chem Biol
1997, 4:513-517.
43. Nesbitt S, Hegg LA, Fedor MJ: An unusual pH-independent and
metal-ion-independent mechanism for hairpin ribozyme catalysis.
Chem Biol 1997, 4:619-630.
44
Young KJ, Gill F, Grasby JA: Metal ions play a passive role in the
hairpin ribozyme-catalysed reaction. Nucleic Acids Res 1997,
25:3760-3766.
45. Earnshaw DJ, Gait MJ: Hairpin ribozyme cleavage catalyzed by
aminoglycoside antibiotics and the polyamine spermine in the
absence of metal ions. Nucleic Acids Res 1998, 26:5551-5561.
46. Murray JB, Seyhan AA, Walter NG, Burk JM, Scott WG: The
hammerhead, hairpin and VS ribozymes are catalytically proficient
in monovalent cations alone. Chem Biol 1998, 5:587-595.
47.
Derrick WB, Greef CH, Caruthers MH, Uhlenbeck OC:
Hammerhead cleavage of the phosphorodithioate linkage.
Biochemistry 2000, 39:4947-4954.
Differences among mechanisms of ribozyme-catalyzed reactions
Masaki Warashina*†, Yasuomi Takagi†, Wojciech J Stec‡ and
Kazunari Taira*†
The catalytic properties of ribozymes depend on the
sophisticated structures of the respective ribozyme–substrate
complexes. Although it has been suggested that ribozymemediated cleavage of RNA occurs via a rather strictly defined
mechanism, recent findings have clearly demonstrated the
diversity of reaction mechanisms.
Addresses
*Department of Chemistry and Biotechnology, Graduate School of
Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
† National Institute for Advanced Interdisciplinary Research, AIST, MITI,
Tsukuba Science City 305-8562, Japan
‡ Polish Academy of Science, Centre of Molecular and Macromolecular
Studies, Department of Bioorganic Chemistry, Sienkiewicza 112,
90-363 Lodz, Poland
Correspondence: Kazunari Taira; e-mail: [email protected]
Current Opinion in Biotechnology 2000, 11:354–362
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
GS
ground state
HDV
hepatitis delta virus
TS
transition state
must be metalloenzymes that require divalent metal ions for
catalysis and that the cleavage mechanism is unique to
ribozymes but common to all types of ribozyme. Recent
advances, however, have revealed examples of metal-ionindependent cleavage by hairpin ribozymes and it has
become clear that multiple mechanisms for cleavage by
ribozymes must exist, depending upon the architecture of
each ribozyme. It has also become clear that the architecture
of HDV ribozyme–substrate complexes can influence the
pKa of the ring nitrogens of cytosine and adenine, with the
resultant perturbed ring-nitrogens being directly involved in
acid/base catalysis [6,7•,8••]. Nevertheless, there is no doubt
that RNA catalysts lacking functional substituents under
physiological conditions exploit metal ions for an appropriate structure and for catalytic function and that the majority
of ribozymes use metal ions as cofactors. The extensive
potential of RNA molecules to act as catalysts and the
sequential events that occur in reactions catalyzed by
ribozymes have been of great interest, and the role of the
catalytic metal ions have been the focus of much experimentation and discussion [9–15,16•,17–19]. Among various
ribozymes studied in the past, we will focus on a few
ribozymes whose enzymatic mechanism became unveiled
over the past year at the atomic level.
Introduction
Catalytic RNAs are called ribozymes and they include
hammerhead, hairpin and hepatitis delta virus (HDV)
ribozymes, group I and II introns, the RNA subunit of
RNase P, and ribosomal RNA [1–5]. Among these
ribozymes, the first two ribozymes to be discovered, by
Altman and Cech, respectively, were the RNA subunit of
RNase P and a group I intron [1–3]. Within the following
five years, small ribozymes, such as hammerhead, hairpin
and HDV ribozymes, were discovered in studies of the
replication, via a rolling-circle mechanism, of certain
viroids, satellite RNAs and an RNA virus [1,4]. These
ribozymes catalyze RNA cleavage/ligation reactions. In the
case of the ribozyme-mediated cleavage of RNA, first a
nucleophile attacks the phosphorus atom at the scissile
phosphate with resultant formation of a pentavalent phosphorus species, which is either a transition state or a
short-lived intermediate, then a leaving oxygen departs
from the phosphorus. The nucleophile can either be an
internal 2′-oxygen in small ribozymes, such as hammerhead, hairpin, and HDV ribozymes, or an external
nucleophile in larger ribozymes, such as the group I intron
and the RNA subunit of RNase P.
Ribozymes are considered to be fossil molecules that originated in a hypothetical prebiotic RNA world. A full
understanding of their mechanisms of action should
enhance our understanding of the evolution of primitive
organisms [1–5]. It has long been assumed that all ribozymes
Reactions catalyzed by the Tetrahymena
ribozyme: a double-metal-ion mechanism
The reaction mechanism of ribozymes that catalyze the
cleavage of RNA phosphodiester linkages, in particular
that of large ribozymes such as group I introns and the catalytic RNA subunit of RNase P, involves external
nucleophiles. By contrast, small ribozymes, such as hammerheads, hairpins and HDV ribozymes, use an internal
nucleophile, namely, the 2′-oxygen of a ribose moiety at
the cleavage site, with the resultant formation of a 3′-terminal 2′-O,3′-O-cyclic phosphate. Despite the large size of
the group I ribozyme of Tetrahymena with its sophisticated
higher-order structure, the extensive efforts made over the
past decade have revealed the details of the mechanism of
the Tetrahymena ribozyme-mediated cleavage of RNAs.
Detailed kinetic and thermodynamic analyses of wild-type
and mutated forms of this ribozyme have helped to define
the reaction mechanism at the atomic level [1,20,21,22••].
Chemical modification at the atomic level has generally
involved introduction of a sulfur atom to replace an oxygen
atom that has the potential to interact with a catalytically
important metal ion. The reduction in the cleavage rate in the
presence of Mg2+ after such a modification (the so-called thio
effect) and the subsequent observation of cleavage with the
phosphorothioate-modified substrate or ribozyme at the same
rate as that of cleavage with the normal RNA components in
the presence of Mn2+ (the so-called manganese rescue effect)
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 1
5′
Exon
G
U (–1) 5′
O
O
3'
Mg 2+
A
Intron
H
–O
pro-Sp
Mg 2+
C
O
2′
G
Ribozyme
2'
O
H
O
3′
P
O
Substrate
A
O
O
3′
O
2′
pro-Rp
O
3′
O
2+
Mg
B
OH
OH
Intron
3′
Current Opinion in Biotechnology
The transition state during the splicing reaction mediated by a
Tetrahymena group I intron. Replacement of a specific oxygen atom
with a sulfur atom followed by metal-dependent cleavage, revealed
three important metal-ion binding sites (A, B and C).
have been taken as evidence that supports the direct coordination of the oxygen atom in question with a metal ion. This
phenomenon can be explained by the hard–soft acid base
(HSAB) rule. According to this rule, a ‘hard acid’, such as
Mg2+, prefers to bind to a ‘hard base’ oxygen atom rather than
to a ‘soft base’ sulfur atom. By contrast, a ‘soft acid’, such as
Cd2+, prefers to bind to a ‘soft base’ sulfur atom. Mn2+ is softer than Mg2+ and, thus, can bind to a soft sulfur atom (as well
as to a hard oxygen atom) and is believed to be the origin of
the manganese rescue effect. Analysis of both the thio effect
and the manganese rescue effect has contributed significantly to our understanding of the mechanism of action of the
Tetrahymena ribozyme. Such analysis has revealed the three
metal-binding sites that are shown in Figure 1 [22••]. Thus, it
is generally accepted that the Tetrahymena ribozyme is a metalloenzyme that operates via the double-metal-ion
mechanism of catalysis, in which Mg2+ at the B site enhances
the deprotonation of the 3′-OH of the guanosine nucleophile
and Mg2+ at the A site stabilizes the leaving 3′-bridging oxygen of U(–1) in the transition state (TS). A third Mg2+ is
postulated to bind at the C site to interact directly with the
2′-OH of the guanosine nucleophile.
Although the above mechanism is, indeed, generally accepted, a linear free-energy analysis of the cleavage of
oligonucleotide substrates with a series of 2′-substituents at
U(–1) suggested that the effect on the catalytic rate of the
355
2′-OH group is larger than might be expected from simple
inductive effects. Specifically, the weaker electron-withdrawing 2′-OH group enhances the chemical cleavage step to
a greater extent than did the stronger electron-withdrawing
2′-F atom of the corresponding 2′-deoxy-2′-fluoro derivative.
Therefore, the possibility of a symmetrical TS, in which the
2′-OH of U(–1) might or might not interact with a metal ion
(as observed at site C), was recently examined [22••].
Despite the absence of lone-pair electrons at the 2′-NH3+
group that might interact with a metal ion, the higher reactivity of the substrate with a 2′-deoxy-2′-NH3+ group than
that of the substrate with a 2′-OH group at U(–1) suggested
that interaction of a metal ion with the 2′-OH of U(–1) might
not be important for catalysis by the Tetrahymena ribozyme.
The higher reactivity of the derivative suggests that donation
of a hydrogen bond from the neighboring 2′-group to the
3′-bridging oxygen might allow specific stabilization of the
TS relative to the ground state (GS), thereby facilitating the
chemical cleavage step. The network of interactions
between the 2′-OH of U(–1), the 2′-OH of A207 and the exocyclic amino group of G22 have been referred to as a catalytic
triad. However, the observation that the chemical cleavage
step with the substrate with a 2′-NH3+ moiety is faster than
that with the 2′-OH group, despite the absence of lone-pair
electrons at the 2′-NH3+ group that can accept a hydrogen
bond from A207, suggests another possibility for the arrangement of active-site groups within this network [22••].
Even though the ribozyme-mediated chemical cleavage
step for the substrate with a 2′-OH group is significantly
(103-fold) faster than that with 2′-H at U(–1), with metalbinding site A being occupied by a Mn2+, the rate constants
for reactions with 3′-deoxy-3′-thio-modified substrates are
similar irrespective of whether there is a 2′-OH group or
2′-H at U(–1). Furthermore, in the presence of Mg2+, with
metal-binding site A unoccupied by a metal ion, the rate
constants for reactions with 3′-deoxy-3′-thio-modified substrates are similar with a 2′-OH group or with 2′-H at U(–1),
indicating that the 2′-OH group at U(–1) does not contribute significantly to the chemical cleavage of the
phosphorus–sulfur bond with a 3′-mercapto leaving group.
Since sulfur is a weaker acceptor of a hydrogen-bond than
oxygen and, furthermore, since sulfur is a significantly better leaving group than oxygen, the 3′-mercapto leaving
group suppresses the catalytic advantage provided by a
hydrogen bond from the 2′-OH in the native TS [22••].
Analysis of the thio effect and rescue experiments have
contributed significantly to elucidation of the mechanism
of action of the large group I ribozyme of Tetrahymena. All
the available data appear to support the refined doublemetal-ion mechanism of catalysis that is shown in Figure 1.
Thio effects and rescue experiments in
reactions catalyzed by hammerhead ribozymes
Metal-ion rescue experiments have also provided considerable information about the sites at which functional
metal ions bind in reactions catalyzed by hammerhead
356
Protein technologies and commercial enzymes
Figure 2
(a)
(b)
Ground state
Transition state
KD
101
[Ca 2+] = 100 mM
100
}
k cat
O
With metal ion
Ca
O
P
O
C17
Ca
Cd
k obs (min -1)
O
H
O (S)
P
P9
O
O
X
[R·M] ‡
R·M
Cd
10-2
C17
O
O
H
O (S)
O
10-1
O
k cleav(PO) = k cleav(PS)
Cd
P9
Native substrate
K GS(PO) = KGS(PS)
K TS(PO) , K TS(PS)
10-3
Phosphorothioate
substrate
10-4
O
Without metal ion
-5
10
(0)
10-8
10-7
10-6
10-5
10-4
10-3
10-2 10-1
[CdCl 2] (M)
C17
O
k (PO) = k (PS)
O
O
O
H
O P O (S)
O
R
C17
O
O
H
O (S)
P
O
R‡
Current Opinion in Biotechnology
Evidence for the identical affinity of a Cd2+ for the native substrate and a
phosphorothioate substrate. (a) Titration with Cd2+ of the ribozymecatalyzed cleavage of each substrate (native substrate, solid line;
phosphorothioate substrate, dashed line) with 100 mM Ca2+ as
background (50 mM MES-Na [pH 6], 37°C). The black arrows indicate
the apparent KD for each ribozyme–substrate complex (left).
(b) Thermodynamic cycles for the rescue of cleavage of the native and/or
the phosphorothioate substrate by addition of Cd2+. Binding of a Cd2+ to
the Rp position of the scissile phosphate in the scheme proposed by
other groups [24,25•] is unlikely. ‘Without metal ion’ means that the
cleavage site is empty but other metal-binding sites are occupied by
background metal ions (right). KGS and KTS are actual KD in the ground
state and the transition state, respectively. KGS(PO) = KGS(PS) can be
estimated from the apparent KD values. The relation of k(PO) = k(PS) was
demonstrated even in an inactivated hammerhead-ribozyme–substrate
complex [47]. R, ribozyme–substrate complex; M, metal ion.
ribozymes [9,23,24,25•,26••]. In such experiments, analogs
of hammerhead ribozymes with phosphorothioate modifications were synthesized, with a sulfur atom replacing
either a bridging or a non-bridging oxygen at the internucleotide phosphorus atom at a preselected position. In
some cases, such substitution resulted in the loss of activity in the presence of Mg2+, which is generally accepted as
the natural metal cofactor. Addition of even trace amounts
of either Mn2+ or Cd2+, however, restored catalysis. These
results supported the hypothesis that the cleavage process
requires direct coordination of a Mg2+ with the phosphate
oxygen and in the case of phosphorothioate analogs
involvement of interaction of a thiophilic metal ion with
the sulfur atom. Specifically, in the presence of Mg2+ or
Ca2+ ions, the rate of the ribozyme-catalyzed cleavage of a
phosphorothioate substrate, in which one of the non-bridging oxygens (the pro-Rp oxygen atom) had been replaced
by sulfur, was nearly four orders of magnitude lower than
that of the reaction with the natural substrate (thio effect).
However, the rates of the ribozyme-catalyzed reactions
with the natural substrate and the phosphorothioate substrate were enhanced by Cd2+ (Cd2+ rescue). Such results
have generally been considered to provide evidence of the
direct interaction of the sulfur atom at the Rp position of
the cleavage site with the added Cd2+; however, that conclusion has been controversial. Results from our laboratory
have indicated that the added Cd2+ binds at the P9 site and
that, contrary to the conclusion reached by other investigators, Cd2+ does not interact with the sulfur atom at the Rp
position of the scissile phosphate in the GS or in the
TS [26••]. Our intepretation is based on the analysis shown
in Figure 2, as discussed below.
We investigated the effects of Cd2+ against a background
of Ca2+. In the case of cleavage of unmodified and phosphorothioate-modified substrates, each curve of the
logarithm of activity versus the logarithm of the concentration of Cd2+ can be divided into three regions. In the
absence of Cd2+, the ribozyme–substrate complex
remains in a less active state in the presence of background Ca2+. Upon addition of Cd2+, Cd2+ replaces
pre-existing Ca2+ within the ribozyme–substrate complex
and, in proportion to the extent of such replacement, the
activity of the ribozyme increases linearly until the relevant Ca2+ has been completely replaced by Cd2+. After
the completion of the replacement of Ca2+ by Cd2+(at
Cd2+ concentrations greater than the apparent KD of
~3 mM; Figure 2, left), the ribozyme–substrate complex
enters the activated state with bound Cd2+.
From the titration curves in Figure 2, an estimate can be
made of respective kinetic and thermodynamic parameters
in the thermodynamic cycle, as follows. As the pre-existing
Ca2+ within the ribozyme–substrate complex was replaced
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
357
by Cd2+ with an identical apparent KD for both normal (PO)
and phosphorothioate (PS) substrates, the binding affinity of
the added Cd2+ to each respective complex in the GS should
be the same: KGS(PO) = KGS(PS). The rate of the cleavage in
the activated complex [R•M]‡ is 1 min–1 at saturating Cd2+
and is the same for both the natural and phosphorothioate
substrates: kcleav(PO) = kcleav(PS). Furthermore, the rate of
nonenzymatic cleavage of the P–O bond in the two substrates is similar: k(PO) = k(PS).
hydroxide that acts as a general base catalyst. Discovery of
this phenomenon provided the first direct demonstration
that a nucleoside can act as an acid–base catalyst in RNA.
As a result, as shown in Figure 3a, the curve showing the
dependence on pH of the self-cleavage of the precursor
HDV genomic ribozyme has a slope of unity at pH values
below 7 (the activity increases linearly as pH increases
with a slope of +1) and then, at higher pH values, the
observed rate constant is insensitive to pH.
The application of thermodynamics and TS theory permits
the calculation of KTS(PO) and KTS(PS). Because three out
of four parameters were the same for the natural substrate
and the phosphorothioate substrate — KGS(PO) = KGS(PS),
kcleav(PO) = kcleav(PS) and k(PO) = k(PS) — the remaining parameter should also be the same for the two substrates:
KTS(PO) = KTS(PS). It is apparent, therefore, that the affinity of Cd2+ for the ribozyme–substrate complex is the same
for both complexes not only in the GS, KGS(PO) = KGS(PS),
but also in the TS, KTS(PO) = KTS(PS), irrespective of
whether the cleavage site includes a regular phosphate or a
modified phosphorothioate moiety.
The slope of unity at low pH is consistent with an increase,
with pH, in the concentration of the metal hydroxide,
which acts as the general base upon deprotonation
(Figure 3b), and a constant amount of the functional protonated form of C75, which acts as the general acid
(Figure 3c). The slope of zero from pH 7 to pH 9 indicates
that the concentration of the metal hydroxide [B:] increases, whereas the concentration of the protonated C75 [AH]
decreases by the same amount [8••]. In agreement with
this interpretation, when C75 was replaced by uracil, the
resultant C75→U mutant, which was unable to assist in the
transfer of a proton, did indeed lack catalytic activity. The
activity of the C75→U mutant could be restored, however,
by the addition of imidazole whose protonated form (imidazolium ion) is known to act as a good proton donor
[7•,8••]. Another mutant, C75→A, in which the ring nitrogen N1 at A75 has a slightly lower pKa than the
corresponding ring nitrogen N3 of C75, supported selfcleavage activity, albeit with reduced efficiency. The
observed pKa of the C75→A mutant was slightly lower
than that of the wild-type C75 ribozyme, supporting the
interpretation that a nucleic acid base acts as a catalyst in
HDV ribozyme-catalyzed reactions as shown in Figure 3.
We must conclude that Cd2+ does not interact directly with
the sulfur atom at the Rp position of the scissile phosphate
either but binds to the P9/G10.1 motif to restore the catalytic activity of the cleavage site [25•,26••]. Consequently,
observations of the thio effect and manganese rescue effect
themselves may not provide decisive arguments in support
of the hypothesis that direct coordination of a metal ion
with the pro-Rp oxygen at the cleavage site is a precondition for hammerhead ribozyme-catalyzed reactions [9,26••].
Even a single substitution of sulfur for oxygen has serious
steric effects, rather than the expected kinetic thio effect,
in reactions that are catalyzed by RNase P [27•]. With a
3′-deoxy-3′-S-phosphorothiolate-modified precursor tRNA,
which can be processed by RNase P, the cleavage site shifts
from the expected phosphorothiolate-modified site to the
adjacent unmodified phosphodiester linkage in the 5′
direction. The bulkiness of the sulfur atom might prevent
the correct positioning of the scissile bond at the active site.
Hepatitus delta virus ribozyme-catalyzed
reactions
Recent reports by three groups have revolutionized our
understanding of the mechanism of HDV ribozyme-catalyzed reactions [6,7•,8••]. For the cleavage of
phosphodiester bonds, the nucleophile must be deprotonated and the leaving group must be protonated or
stabilized by a metal ion. As shown in Figure 1, the developing negative charges in the TS in the Tetrahymena
ribozyme-catalyzed reaction are stabilized by direct interactions with metal ions. The novel finding with respect to
the mechanism of catalysis by the HDV ribozyme is that
the pKa of C75 is perturbed to neutrality in the
ribozyme–substrate complex and, more importantly, C75
acts as a general acid catalyst in combination with a metal
Further convincing evidence for this model comes from
the observation that, in the absence of divalent metal ions
(in the absence of base B; Figure 3b) and in the presence
of a high concentration of monovalent cations (~2 M NaCl
and 1 mM EDTA), the logarithm of the observed rate constant decreased with pH with a slope of –1 (as shown in
Figure 3c) with only general acid catalysis operative (in the
absence of the metal hydroxide as a general base). This
type of profile clearly demonstrates that the observed pKa
for self-cleavage results from the general acid rather than
from the general base. It is noteworthy that [Co(NH3)6]3+
which is known to be a substitutionally inert transition
metal complex (kex < 10–10 s–1 even at acidic pH) inhibited the Mg2+-catalyzed reaction in a competitive fashion,
suggesting that [Co(NH3)6]3+ might bind to the same site
as the functional Mg2+ with outer-sphere coordination but
does not ionize. The similar rate constants in the presence
of Ca2+ and of Mg2+ are also consistent with the action of a
hydrated metal ion as a Brönsted base rather than a Lewis
acid in the HDV ribozyme.
There was a significant, apparent D2O solvent isotope
effect over the entire range of pH values, confirming that
transfer of a proton occurs in the TS (the ratio of rate
358
Protein technologies and commercial enzymes
Figure 3
pKa = 6
pKa = 11
(d)
5'
log k
5'
O
C21
O
O
O
O
O
P O
H
H N
O
C22
H+
0.7
O H OH
P O
Mg
H
O
G-1
H
N
C75 O
N
3'
U-1
a
=
1
O H
Mg 2+
H
NH2
pKa = 11
H
:
H
O
H
O
Mg 2+
0.6
3'
(b)
:
O
O
pH
log [B:]
pK
1.4
pK a
(a)
a
pK
0.5
H
=
6.1
N
Mg 2+
N
O
C75
NH2
O
N
N
H
O
C75
Mg 2+
0.4
0
5
10
15
pK a
(e)
pH
3.5-fold
pKa = 6
NH2
log [AH]
6.5-fold
log k
(c)
8.5-fold
N
N
C75
H
O
NH2
N
O
N
H2O
D2O
C75
5
pH
10
pL
Current Opinion in Biotechnology
Kinetic analysis of HDV ribozyme-catalyzed reactions. (a) A schematic
replotting of the pH-rate profile of HDV ribozyme-catalyzed reactions
[8••]. This profile has pKa values of 6 and 11. The solid line and the
dotted line represent the experimentally obtained curve and the
theoretical curve, respectively. This profile can be explained by a
combination of the dependence on pH of the concentration of the active
base [B:], as shown in (b), and of the concentration of the active acid
[AH], as shown in (c). (b) The logarithm of the concentration of a general
base catalyst in an active form is plotted against pH. The pKa is 11. (c)
The logarithm of the concentration of a general acid catalyst in an active
form is plotted against pH. The pKa is 6. (d) Isotope effects on the
acidities of phenols and alcohols as a function of their acid strength, pKa
(for details, see [9,28,29]). (e) Theoretical curves for the activity of the
HDV ribozyme in H2O and D2O. The pKa values in H2O and in D2O for
C75 of 6.1 and 6.5, respectively, were taken from the experimental data
[8••]. Using the pKa in H2O for a magnesium-bound water molecule as
being 11.4, we calculated the corresponding pKa values in D2O, from
the linear plot in (d), to be 12 [9]. Theoretical curves were plotted on the
assumption that the value of the intrinsic isotope effect was 2. The
following equation was used to plot the graph of pL versus log(rate):
log kobs = log kmax – log (1 + 10[pKa(base) – pL]) – log (1 + 10[pL – pKa(acid)]) –
log α. Where kmax is the rate constant in the case of all acid and base
catalysts in active forms, and α is the coefficient of the intrinsic isotope
effect, that is, kmax(H O)/kmax(L O). In the pH profile, α = 1; in the pD profile,
2
2
α = 2 under the above mentioned assumption.
constants, kcat(H O):kcat(D O), being as high as 10; see
2
2
[8••]). Moreover, because, firstly, the observed pKa for
self-cleavage is that for the general acid and secondly,
the overall rate-limiting step appears to be the cleavage
of the bond between the phosphorus and the 5′-leaving
oxygen (see below and Figure 4), the transferred proton
in the TS must be that of C75. As the concentration of
the protonated C75 in H2O can be higher than that in
D2O at a fixed pH, the difference in pKa (∆pKa), which
equals log(KaH2O/KaD2O), must be taken into account
when the D2O solvent isotope effect is calculated
[9,28,29]. It is possible to estimate the relative concentrations of the active species in H2O and in D2O because
a linear relationship exists between them, as shown in
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 3d [9]. Under the conditions of measurements,
the pKa of C75 is 6.1 and, thus, the estimated ∆pKa turns
out to be 0.53, as indicated by the dashed arrow in
Figure 3d. Indeed, in the pD-logk profile, the pKa is
shifted upward by ~0.4 ± 0.1 pH units, consistent with
the estimated value and with proton transfer by a ring
nitrogen [8••]. If we take the pKa of C75 as 6.1 in H2O
and 6.5 in D2O, and the pKa values for magnesiumbound water to be 11.4 and 12.0, respectively, and if we
assume that the value of the intrinsic D2O solvent isotope effect is two, we can generate theoretical curves for
HDV-catalyzed reactions in H2O and D2O, as shown in
Figure 3e. The agreement of our theoretical curves with
the observed curves [8••] confirms that proton transfer
does indeed occur from protonated C75 in the P–(5′–O)
bond-cleaving TS. This conclusion is different from the
case of hammerhead ribozymes because, in that case, the
observed isotope effect (k cat(H O)/k cat(D O) = 4.4; see
2
2
[9,28,29]) can be fully explained by the difference in relative concentrations of the active species in H2O and in
D2O, with an intrinsic D2O solvent isotope effect being
zero [9,28,29].
Thus, in the case of HDV ribozyme-catalyzed reactions, all
the available data support the reaction mechanism shown
in Figure 3a. The donation of a proton is favored by this
model, which involves an acid with an optimal pKa of 7.
The ability of the HDV ribozyme to perform general
acid–base catalysis effectively under physiological conditions appears to be unique among known ribozymes.
Importance of acid catalysis in reactions
catalyzed by hammerhead and HDV ribozymes
Nonenzymatic hydrolysis, as well as small-ribozyme-mediated hydrolysis, of RNAs occurs via nucleophilic attack by
the 2′-oxygen that generates the pentacoordinate intermediate or TS (via the first putative TS [TS1]). Such attack is
followed by the cleavage of the bond between the phosphorus and the 5′-oxygen leaving group (via the second
putative TS [TS2]; Figure 4). Among the two putative
TSs, TS2 is the overall rate-limiting TS (attack by the
2′-OH on the phosphorus atom is easier than cleavage of
the P–O[5′] bond and, thus, TS2 always has higher energy
than TS1). This conclusion was confirmed in experiments
with an RNA analog with a 5′-mercapto leaving group. If
the formation of the intermediate were the rate-limiting
step (i.e. if TS1 were to be a higher-energy state than
TS2), the phosphorothiolate substrate should be
hydrolyzed at a rate similar to the rate of the hydrolysis of
the natural RNA, because the 5′-bridging phosphorothiolate linkage would not be expected to enhance the attack
by the 2′-oxygen. In contrast, if the decomposition of the
intermediate were the rate-limiting step (i.e. if TS2 were a
higher-energy state than TS1), we would expect that the
phosphorothiolate substrate would be hydrolyzed much
more rapidly than the natural RNA because the pKa of a
thiol is more than 5 units lower than that of the corresponding alcohol. Several groups have confirmed that the
359
phosphorothiolate-modified substrate is significantly more
reactive than the natural RNA in nonenzymatic hydrolytic
reactions [9,10] and, thus, TS2 is indeed always a higherenergy state than TS1 in the cleavage of natural RNAs.
In the case of the hammerhead-ribozyme-catalyzed reactions, the RNA substrate with a 5′-mercapto leaving group
is extremely reactive and handling is difficult [30,31]. In our
laboratory, however, we recently succeeded in measuring
rate constants of 5′-phosphorothiolate-modified and natural
substrates under controlled conditions (measurements
were performed under conditions such that curves of pH
versus log[rate] had a slope of unity for both substrates, and
all undesired side reactions could also be avoided; Y Takagi
and K Taira, unpublished data). As the 5′-phosphorothiolate analog of the substrate was hydrolyzed by hammerhead
ribozymes at significantly higher rates than those measured
with the natural RNA substrate, in agreement with our previous conclusion [31], TS2 must also be a higher-energy
state than TS1 in these ribozyme-catalyzed reactions.
Because the overall transition-state structure in the hydrolysis of RNA is TS2, regardless of whether the reaction
proceeds via a concerted one-step mechanism or via a twostep mechanism with a stable pentacoordinate
intermediate, it is important that any enzyme that catalyzes
the hydrolysis of RNA should stabilize TS2 by donating a
proton to the 5′-leaving oxygen or by ensuring coordination
of a metal ion to the leaving oxygen.
The above scenario should also be applicable to HDVribozyme-catalyzed reactions and, thus, the removal of the
possibility of acid catalysis in the C75→U mutant suppressed the enzymatic activity. By contrast, self-cleavage
of the HDV ribozyme in the absence of divalent cations,
but in the presence of high concentrations of monovalent
cations, suggests that, at high concentrations, monovalent
cations can replace divalent cations in the tertiary folding
of the RNA. Thus, divalent cations are not absolutely
essential for the folding or cleavage activity of the HDV
ribozyme, although a functional Mg2+ does participate in
the cleavage process under physiological conditions.
Metal ion involvement in hammerhead
ribozyme-catalyzed reactions
Base catalysis mediated by Mg2+-hydroxide was postulated as
the mechanism of the chemical cleavage step in reactions catalyzed by a hammerhead ribozyme on the basis of the original
profiles of pH versus the rates of various metal ion-catalyzed
reactions of hammerhead ribozymes [32]. The single-metalion mechanism, in which Mg2+-hydroxide acts as a general
base and abstracts a proton from the 2′-OH internal nucleophile [32], is supported by the finding that no metal ion was
found close to the 5′-leaving oxygen in the crystallographic
structure of a freeze-trapped conformational intermediate of a
hammerhead ribozyme [33,34]. In the more recently determined crystal structure of a modified hammerhead ribozyme
[35], however, Co2+ was located close to the 5′ oxygen atom of
the scissile phosphate. Based upon the crystallographic
360
Protein technologies and commercial enzymes
Figure 4
5′
5′
C21
TS2
O
O
O P
O H
H
N
O
H
N
C22
C75
5′
O
O P
O P
O
H
O H
N
O
H
N
C22
Relative energy
C75
N
3′
O
O
P
O
O
R
H
O
Mg
H
2
G1
O H
3′
O
TS2
G1
5′
O H
O
OH
O−
O
Mg
O
G1
P
O
H N H
TS1
O H
C75
N
R
P O
H
H N
O
H
N
C22
C75 O
O
P
O
N
+
O
3′
O H
H
O
H
Mg
2
HO
O
O
O
O
+
O
U-1
O
3′
k1
U-1
O
O
O
O
5′
3′
O H OH
O
Mg
O
R
C21
k2
O
O
O
TS1
O
5′
U-1
O
C21
N
3′
5′
U-1
O
G1
O H
3′
k1
P(V)
k2
k-1
N
R
Current Opinion in Biotechnology
Energy diagram for cleavage by a ribozyme (HDV ribozyme) of its substrate. The rate-limiting step of the reaction with the normal substrate is the
cleavage of the P-(5′-O) bond. The transition state structures of TS1 and TS2 are also shown.
structure, molecular dynamic simulations led to the proposal
of a new and alternative single-metal-ion mechanism. In the
proposed mechanism, one Mg2+ coordinates directly and
simultaneously to the pro-Rp oxygen and the adjacent oxygen
of the attacking hydroxyl group at the cleavage site, acting as
a Lewis acid to enhance the attack by the 2′-OH moiety on
the phosphorus atom. In addition, it was also proposed that
one of the outer-sphere water molecules that surround the
metal ion is located such that it might act as a general acid to
donate a proton to the 5′-leaving group [36]. As mentioned
above, however, three independent groups recently found no
evidence for the direct coordination of a metal ion to the proRp oxygen, at least in the GS [25•,26••,37]. It was also noted
that the absence of such an interaction could explain the
absence of a thio effect in the hammerhead-catalyzed cleavage of a phosphorodithioate linkage [37]. Our analysis,
depicted in Figure 2, also did not support such an interaction
in the TS [26••].
In contradiction, several reports have now appeared that provide experimental evidence for a double-metal-ion
mechanism of catalysis in hammerhead-catalyzed reactions.
Studies on solvent isotope effects and kinetic analysis of a
modified substrate with a 5′-mercapto leaving group at the
cleavage site have lent strong support to the double-metalion mechanism of catalysis [28,31]. Furthermore,
hammerhead-ribozyme-mediated cleavage has been analyzed as a function of the concentration of La3+ ions in the
presence of a fixed concentration of Mg2+ ions. In this way,
the role of lanthanum could be monitored and the metal ions
could be identified that were directly involved in the cleavage reaction [38,39]. The resultant bell-shaped curve that
originated from activation by the 5′-oxygen-bound La3+ and
deactivation by the 2′-oxygen-bound La3+, shown in
Figure 5, was used to support the proposed double-metal-ion
mechanism of catalysis. Other studies, however, demonstrated that binding of a metal ion (the P9 metal ion) to the
Differences among mechanisms of ribozyme-catalyzed reactions Warashina et al.
Figure 5
C
O
Activity of ribozyme
O
O
O
P
Mg 2+ O
O
O–
O
C
O
Mg2+
O
C
O
O
3+
O
3+
La
O
P
O
O
P
Mg 2+
La
O
3+
La
O–
O
O–
O
LaCl3 (µM)
Current Opinion in Biotechnology
A bell-shaped titration curve for cleavage by hammerhead
ribozymes. Both the wild-type ribozyme and the ribozyme with a
7-deaza group at G10.1 generated the same bell-shaped titration
curve (for details, see [38,39,40•]).
pro-Rp oxygen (P9 oxygen) of the phosphate moiety of
nucleotide A9 and to N7 of nucleotide G10.1 is critical for
efficient catalysis, despite the considerable distance (~20 Å)
between the P9 metal ion and the phosphorus of the scissile
phosphodiester group in the GS. In fact, it was demonstrated that an added Cd2+ binds first to the pro-Rp phosphoryl
P9 oxygen and not to the pro-Rp phosphoryl oxygen at the
cleavage site [25•,26••]. Therefore, it was difficult to exclude
completely the possibility that La3+ ions might have replaced
the P9 metal ion and, as a result, might have created conditions represented by the bell-shaped curve. In order to clarify
this situation, we examined a modified hammerhead
ribozyme that included an N7-deazaguanine residue instead
of G10.1. Because the data from our experiments with the 7deaza-ribozyme were free from potential artifacts that might
have resulted from the binding of a La3+ to N7 at G10.1, the
bell-shaped curve obtained for 7-deaza-ribozyme upon the
addition of La3+ to a reaction mixture that contained Mg2+,
as reproduced in Figure 4, strongly supports the proposal that
a double-metal-ion mechanism operates in the cleavage reactions catalyzed by hammerhead ribozymes [40•]. Recent
ab initio molecular orbital calculations also support the double-metal-ion mechanism [41].
Conclusions: diversity among the mechanisms
of ribozyme-catalyzed reactions
It has been well established that the Tetrahymena ribozyme is
a metalloenzyme that operates according to the doublemetal-ion mechanism of catalysis. In general, as indicated
above, other ribozymes also require metal ions for catalysis.
Metal ions are almost certainly needed to induce the folding
of each ribozyme into its active conformation, and the folded
ribozyme might recruit metal ions at correct positions for
direct participation in the chemical cleavage process, as in the
case of the Tetrahymena ribozyme. Alternatively, the folded
RNA structure might itself play a role in the cleavage
361
mechanism [16•,37], as in the case of the HDV ribozyme. In
this latter ribozyme, the folded RNA perturbs the pKa of C75
and, as a result, C75 can perform effective general-acid catalysis under physiological conditions. Evidence that the folded
RNA structure itself functions in the cleavage mechanism
comes from studies of hairpin ribozymes. Three independent
groups have demonstrated that the hairpin ribozyme is almost
fully functional when [Co(NH3)6]3+ replaces Mg2+ [42–44].
The studies of cleavage induced by the exchange-inert
[Co(NH3)6]3+ complex have been extended to include nonmetallic compounds, and hairpin ribozyme-mediated
cleavage has been induced both by aminoglycoside antibiotics and by polyamines [45]. The mechanism of catalysis of
the simplest and most extensively studied hammerhead
ribozyme remains controversial. The activities of hammerhead ribozymes under physiological conditions apparently
depend on Mg2+. Nevertheless, high concentrations of monovalent metal ions (1–4 M monovalent cations, such as Li+,
Na+, and NH4+) can perform the functions of divalent metal
ions [46]. Furthermore, the cleavage activity of hammerhead
ribozymes in the absence of divalent cations suggests that
divalent cations are not absolutely essential for folding or
cleavage in hammerhead ribozyme-catalyzed reactions, as is
also the case for the HDV ribozyme. It remains to be determined whether such high concentrations of monovalent
metal ions might stabilize the substantial negative charge that
develops on the 5′-oxygen atom of the leaving group in hammerhead ribozyme-catalyzed reactions.
In conclusion, the evidence that has accumulated over the
past two years clearly demonstrates the diversity of
ribozyme-catalyzed reactions. Nevertheless, acid-base
catalysis appears to operate in every case even if many
details remain to be clarified.
Acknowledgement
M Warashina and Y Takagi contributed equally to the work. WJ Stec thanks
MITI for the financial support that paid for his three-month stay at the
Laboratory of Molecular Biology of the National Institute of Advanced
Interdisciplinary Research in Tsukuba.
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