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mM at 37°C. Aliquots withdrawn at different time intervals were assayed for the residual activity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Aliquots withdrawn at different time intervals were transferred immediately to activity assay so-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at 37°C. Aliquots withdrawn at different time intervals were checked for residual activity as de-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
3
.
1
. Assay of SH groups of urease Free SH groups of freshly isolated urease were
assayed by monitoring its reaction with excess DTNB, spectrophotometrically at 412 nm. The
results are shown in Fig. 1. Note the presence of SH groups of different reactivities. There is almost
a ‘burst’ of absorbance increase initial 60 s, followed by a slower reaction completed in 6 min
and a very slow absorbance increase which is linear with time and is not completed even after 15
min. If a correction is applied for the last category of very slow reacting phase groups by extrapola-
tion to zero time Fig. 1, the absorbance increase in the first two phases corresponds to the reaction
of 5.82 9 0.13, i.e. 6 ‘accessible’ SH groups per hexamer enzyme protein molecule mol. wt. 480
kDa, i.e. one SH group per monomeric subunit.
When the enzyme was denatured by SDS-heat treatment before adding DTNB, there was an
instantaneous increase in absorbance with no fur- ther time-dependent change data not shown. The
absorbance increase corresponds to 12.1 9 0.1, i.e.
12 SH groups per molecule, i.e. about two SH groups per monomeric subunit.
3
.
2
. Kinetics of reaction of DTNB with the
‘
accessible
’
SH groups A semi-log plot of the reaction of accessible SH
groups of urease with DTNB shows biphasic ki- netics Fig. 2A. For this plot, the difference be-
tween the extrapolated value of the slow reaction and the experimentally observed absorbance in-
crease at the corresponding time was taken as indicative of the residual ‘accessible’ SH groups.
The semi-log plot clearly shows that some of the reactive SH groups react faster than the rest fast
and slow phases. The time course of the reaction of the ‘accessible’ SH groups of urease with excess
DTNB can be represented by a rate equation consisting of two first-order terms, corresponding
to the fast and slow phases of the reaction Eq. 1, see Materials and methods. Similar biphasic
kinetics of reaction of urease and DTNB was observed at pH 7.5 data not shown, however the
reaction at this pH was slower. The values of the amplitudes and rate constants at different pH are
shown in Table 1.
When loss of enzyme activity was monitored with DTNB in a time-dependent manner, again
biphasic kinetics was observed Fig. 2B. The time- course of inactivation of pigeonpea urease with
DTNB is consistent with Eq. 2 see Materials and methods. The values of the amplitudes and
rate constants are shown in Table 1.
In these experiments DTNB was always present in large excess, so that the observed kinetic bipha-
sicity cannot be attributed to limited DTNB con- centration. It must, therefore, represent different
reactivities of the various accessible SH groups. It is noteworthy that the amplitudes of the fast and
the slow phases are nearly equal; each phase ac- counts for about half of the total absorbance
changeactivity.
3
.
3
. Effect of SH group modifying reagents on urease acti6ity
Pigeonpea urease was inactivated on incubation with low concentrations of SH reagents, like p-
CMB 100 mM, NEM 250 mM or IAM 5 mM. In each case, the reagent concentration was in
large excess as compared to that of the enzyme 0.75 mgml corresponding to 1.56 mM. A semi-
log plot of the data obtained with p-CMB shows biphasic kinetics of the reaction Fig. 3. The
complete time-course of inactivation of pigeonpea urease with excess p-CMB is consistent with Eq.
2, similar to that given above for the reaction with
DTNB. In
the data
of Fig.
3, A
fast
A
slow
50 of the initial activity. A similar pattern of inactivation is observed
with NEM and IAM data not shown. Values of the amplitudes and the rate constants of the two
phases are given in Table 1. In each case, A
fast
A
slow
half of the initial activity. Inactiva- tion of urease with each reagent is consistent with
the following scheme.
Fig. 1. Kinetics of reaction of pigeonpea urease with DTNB. The reaction of the enzyme 0.75 mgml and DTNB 0.2 mM, was carried out in 50 mM TEA buffer pH 8.5 at 37°C and monitored at 412 nm.
Fig. 2. A Semi-log plot of the data of Fig. 1 for first 8 min, after correcting the very slow reaction see text. B Semi-log plot for the inactivation kinetics of pigeonpea urease acivity with DTNB 0.2 mM at 37°C, pH 8.5.
Active Enzyme
Excess reagent Fast
Half-Active Enzyme
Excess reagent Slow
Inactive Enzyme 3
This is similar to the reaction of accessible SH groups of pigeonpea urease with excess DTNB.
Thus, the reactivity of SH groups with a variety of reagents is suggestive of half-site reactivity.
In a separate set of experiments, the enzyme was incubated with p-CMB 100 mM for 11 min and
then treated with a large excess of freshly prepared cysteine 1 mM. Note that the inactivation by
p-CMB is largely reversed on the treatment with cysteine Fig. 4.
The relationship between the accessible SH groups and catalytic activity of urease has been
investigated in a separate experiment. Pigeonpea
Table 1 Amplitudes and the rate constants for the inactivation of pigeonpea urease with various SH reagents
Fast phase Slow phase
Reagent A
fast
k
fast
min
− 1
A
slow
k
slow
min
− 1
50.9 9 0.5 0.43 9 0.01
49.1 9 0.5 DTNB
a
0.07 9 0.01 pH 7.5, 0.2 mM
a
51.9 9 0.5 2.80 9 0.1
48.1 9 0.5 pH 8.5, 0.2 mM
a
0.12 9 0.01 pH 8.5, 0.2 mM
b
DTNB
b
51.3 9 0.5 2.82 9 0.1
48.7 9 0.5 0.126 9 0.001
p-CMB
b
pH 7.3, 0.100 mM 51.0 9 0.5
0.92 9 0.04 49.0 9 0.5
0.045 9 0.003 51.8 9 0.5
1.15 9 0.1 48.2 9 0.5
pH 7.3, 0.125 mM 0.069 9 0.005
50.8 9 0.5 0.47 9 0.01
49.2 9 0.5 0.046 9 0.003
NEM
b
pH 7.3, 0.250 mM 51.7 9 0.5
0.53 9 0.03 48.3 9 0.5
pH 7.3, 0.330 mM 0.063 9 0.004
51.0 9 0.5 0.43 9 0.01
49.0 9 0.5 0.022 9 0.004
IAM
b
pH 7.3, 5 mM 51.5 9 0.5
0.54 9 0.04 pH 7.3, 8 mM
48.5 9 0.5 0.047 9 0.003
a
Kinetics of reaction of SH groups based on absorbance change at 412 nm.
b
Kinetics of inactivation of the enzyme based on activity measurements.
urease was incubated with excess SH group reagents p-CMB, NEM and IAM and aliquots withdrawn
at different time intervals were assayed for residual activity as well as for the residual accessible SH
groups titration with DTNB as described earlier. A plot of residual activity against the number of
accessible SH groups blocked is shown for p-CMB in Fig. 5. A linear relationship is observed between
the two parameters and all the six ‘accessible’ SH groups need to be blocked for complete inactivation
of the urease.
Urea, at 0.2 M concentration, brought about a weak protection against inactivation by either NEM
or p-CMB. Finally, when urease was incubated with excess NEM in the presence of different concentra-
tions of fluoride ion, the enzyme was protected against time-dependent inactivation Fig. 6.
4. Discussion
