plant enzyme thiols differ in their chemical proper- ties [8,9].
The urease active site is conserved among all sources as shown by extensive identity in the
protein sequences, a common requirement for nickel, and similarity in behavior towards in-
hibitors and inactivating reagents. For example, the presence of a thiol in jack bean urease is
supported by studies showing that alkylating and disulfide reagents inactivate the enzyme [8,10].
Similarly, disulfides inactivate K. aerogenes urease, apparently by disulfide exchange with the active
site cysteine [9,11]. It has been suggested that the slow loss of jack bean urease activity in the pres-
ence of b-mercaptoethanol and oxygen was due to the formation of mixed disulfide involving a
thiol located at the active site [12]. Titration of native jack bean urease has been carried out with
NEM [10,13] and 5,5-dithiobis-2-nitrobenzoate DTNB [10] and a pK
a
value of 9.15 at 25°C has been assigned to the unique cysteine [8]. The
amino acid sequence has been reported for a 34- residue cyanogen bromide peptide, which contains
this residue [14]. In contrast, urease from Staphy- lococcus xylosus has a threonine, instead of cys-
teine, at the active site; it is not inhibited by the SH group inhibitor and its DNA sequence encodes
no cysteine residue at this position [15]. Very recent structural studies on urease from Bacillus
pasteurii have shown that Cys
322
plays a significant role in the catalytic process, even though it is not
essential [16,17]. Such detailed structural informa- tion on both the binuclear Ni centre in the enzyme
active site and the mode of thiol inhibition is vital for structure-based rational design of urease re-
lated drugs [16]. Alkylating reagents specific for thiol groups, like iodoacetate IA, iodoacetamide
IAM, NEM and p-CMB, have been shown to inhibit several microbial ureases thus, the micro-
bial ureases possess a thiol group [1].
Urease from dehusked pigeonpea Cajanus cajan L. seeds has been purified to apparent homogene-
ity, partially characterized [18,19], and shown to be an important enzyme for analytical purposes
[20 – 23]. In continuation of our work on urease characterization from pigeonpea, the present pa-
per describes the significance of sulfhydryl groups in the activity of urease. Relative reactivities of the
functional groups -SH and site – site heterogene- ity within the urease hexamer molecule have also
been described.
2. Materials and methods
2
.
1
. Enzyme Urease was purified from dehusked seeds of
pigeonpea Cajanus cajan L. seeds as described earlier [18]. The isolated enzyme was more than
95 pure as judged by native and SDS-PAGE. The specific activity of the purified enzyme varied
from batch to batch in the range 4500 – 5500 units mg protein.
2
.
2
. Chemicals DTNB, p-CMB, NEM, IAM, Bovine Serum
Albumin BSA and triethanolamine buffer were obtained from Sigma Chemical Co. St. Louis,
MO, USA.. Tris Buffer, urea enzyme grade, trichloroacetic acid TCA and sodium fluoride
were from Sisco Research Labs. Mumbai, India. Nessler’s reagent was procured from HiMedia
Labs. Mumbai, India. All other chemicals were of analytical grade. All solutions were prepared in
triple distilled water from an all-quartz distillation assembly.
2
.
3
. Enzyme and protein determination For routine assay of urease activity, ammonia
liberated in a fixed time interval at an enzyme-sat- urating concentration of urea was determined us-
ing Nessler’s reagent as described earlier [19]. The yellow color produced was measured spectropho-
tometrically at 405 nm. The amount of ammonia liberated in the test solution was calculated by
calibrating the reagent with standard ammonium chloride solution. An enzyme unit has been
defined as the amount of enzyme required to liberate 1 mmole of product ammonia per minute
under our test conditions 0.1 M Urea, 0.05 M Tris-acetate buffer, pH 7.3, 37°C.
Protein was assayed by the method of Lowry et al. [24] with BSA as standard.
2
.
4
. SH group assay Enzyme SH groups were assayed using DTNB
as the reagent [25]. The assay solution 1.05 ml contained 50 mM TEA buffer, pH 8.5, DTNB 0.2
mM and enzyme 0.75 mgml. The enzyme aliquot 0.2 ml was added last and the reaction
monitored at 412 nm
412nm
1.39 × 10
4
and 1.31 × 10
4
M
− 1
cm
− 1
at pH 8.5 and 7.5, respectively.
Simultaneously, in a similar set of experiment pigeonpea urease was incubated with DTNB 0.2
mM at 37°C. Aliquots withdrawn at different time intervals were assayed for the residual activity
of the enzyme.
2
.
5
. Inacti6ation of urease with SH group modifying reagents
The enzyme was incubated with the desired concentration of specified reagent p-CMB, NEM
or IAM in 50 mM Tris-acetate buffer, pH 7.3 at 37°C in the absence or presence of substrate urea.
Aliquots withdrawn at different time intervals were transferred immediately to activity assay so-
lution 2.0 ml, containing 0.9 ml of 50 mM Tris- acetate buffer, pH 7.3 and 1.0 ml of 0.2 M urea.
In a separate set of experiments, the residual SH groups were assayed in the aliquots as described
earlier.
For reactivation studies, the enzyme 0.75 mg ml was incubated with p-CMB 100 mM for 11
min in Tris-acetate buffer, pH 7.3, at 37°C fol- lowed by addition of excess cysteine 1 mM and
the recovery of activity was monitored at different time intervals.
For experiments measuring fluoride ion protec- tion against NEM inactivation, the enzyme was
incubated with NEM 250 mM in the presence of sodium fluoride 500 and 750 mM in assay buffer
at 37°C. Aliquots withdrawn at different time intervals were checked for residual activity as de-
scribed above.
2
.
6
. Analysis of kinetic data The time course of absorbance change at 412
nm and inactivation of the enzyme activity were analyzed according to Eq. 1 and Eq. 2, respec-
tively given below. Initial estimates of the rate constants and amplitudes were obtained from
semi-log plots as described earlier [26]. Their val- ues were refined by iterative curve fitting proce-
dure [27].
DA −
D A
t
=
A
fast·e
− k
fast
·
t
+ A
slow
·e
− k
slow
·
t
, 1
where DA
t
is the corrected absorbance increase at time ‘t’ and DA
the corrected absorbance in- crease when all the accessible SH groups have
reacted with excess DTNB, k
fast
and k
slow
are the pseudo-first order rate constants and A
fast
and A
slow
are amplitudes expressed as absorbance in- crease so that A
fast
+ A
slow
= D
A of the fast and
slow phases, respectively.
A
t =
A
fast·e
− k
fast
·t
+ A
slow
·e
− k
slow
·
t
, 2
where A
t
is the percent residual activity at time ‘t’, A
fast
and A
slow
are the amplitudes expressed as percent of initial activity and k
fast
and k
slow
are the pseudo-first order rate constants of the fast and
the slow phases of reaction, respectively.
3. Results
