A.K. Vashishtha et al. / Archives of Biochemistry and Biophysics 584 (2015) 98e106
105
Table 4
Summary of pKa values for V2/KSaccEt pH-rate profile.
Enzyme
pK1
pK2
pH independent value (Mꢀ1 sꢀ1
)
Fold decrease
WT
D126A
C154S
Y99F
D126A-Y99F
D126A-C154S
7.6 0.1
8.8 0.3
8.1 0.2
7.1 0.4
9.3 0.2
8.5 0.2
9.9 0.2
9.9 0.3
10.3 0.2
9.5 0.3
10.3 0.5
NDa
(2.9 0.5) ꢃ 104
(1.0 0.3) ꢃ 104
2.9
391
2536
337
74
11
8
2
86 22
56
8
518
a
ND is not determined.
Table 5
mutant enzyme exhibits small changes in the rate and pH depen-
dence of the reaction. The groups observed on the basic side of the
Solvent kinetic isotope effects.a
D2
D
2 O
Enzyme
OV2
ðV2=KSacc
Þ
profile are assigned to the
a
-amines of Glu and bound AASA. Thus,
prior to I in Scheme 2, the
a-amine of Glu is likely deprotonated by
WTb
D126A
C154S
Y99F
D126A-C154S
D126A-Y99F
1.8 0.1
2.6 0.2
2.8 0.7
2.8 0.1
2.6 0.1
2.1 0.1
1.8 0.1
1.9 0.6
2.8 0.7
1.9 0.2
an enzyme residue to give I.
In the direction of Sacc formation, V1 decreases at low and high
pH giving pKas of 5.7 and 7.9, and these must reflect the -amines of
the bound forms of Glu and AASA. The pKa of 5.7 is assigned to the
-amine of Glu and that of 7.9 assigned to the -amine of AASA
bound to enzyme. The low value, 5.7, of the pKa assigned to the
amine of Glu likely reflects placement of the -amine into a
a
2.22 0.03
3.1 0.3
a
a
a
Data were obtained in the pH(D) independent region of the pH rate profiles for
V2 and V2/KSacc for each of the mutant enzymes.
a
-
a
b
From Ref. [5].
nonpolar environment, or its close proximity to another positively
charged group within the active site. There is precedence for both
of the above possibilities giving a decrease in the pKa value of pri-
mary amines. Acetoacetate decarboxylase exhibits a pKa of 6 for
Lys115 in the active site as a result of its placement in a nonpolar
environment [12]. In addition, Lys256 in aspartate aminotrans-
ferase also exhibits a pKa of 6 as a result of its proximity to the
amine of pyridoxamine 50-phosphate [13]. Of the two possibilities,
subsequent conformational change that occurs once reactants are
bound. Thus, the effect on binding of NADPH is likely linked to the
decrease in the rate of the pre-catalytic conformational change. A
similar effect is observed (to a somewhat lesser extent for D126A)
on V2/KNADP in the direction of Glu formation, Table 3.
the latter is most likely, given the proximity of the
the -amine of AASA, Fig. 1.
In the direction of Glu formation, V2 decreases at low pH giving a
pKa of 7.2, which reflects the -amine of the AASA moiety of Sacc.
The V2/KSacc decreases at low and high pH, giving pKas of 7.6 and 9.9,
which reflect -amine and secondary amine of Sacc, respectively. If
data were collected to low enough pH in the previous study [5], an
additional pKa of about 5.7 would be expected for D126, which
must accept a proton from the secondary amine (either directly or
via a water molecule bridge).
a-amine of Glu to
3.4. Re-interpretation of the pH-rate profiles for WT saccharopine
reductase [5]
a
a
The pH-rate profiles obtained for WT enzyme were interpreted
in terms of enzyme residues acting as general acidebase catalysts.
On the basis of data presented in this study and the results of the
QMeMM studies published previously [8], the chemical mecha-
nism is auto-catalyzed by the bound reactants and the pH-rate
profiles reflect pKa values of the reactants. Thus, pH-rate profiles
obtained in Ref. [5] will be re-interpreted in terms of Scheme 2,
which outlines the mechanism proposed by Almasi et al. [8]. Given
the proposed ordered kinetic mechanism in which Glu adds last in
the direction of Sacc formation and Sacc adds last in the direction of
Glu formation, the V1, V2, V1/KGlu and V2/KSacc are the parameters of
interest since all include the rate constants for the chemical steps.
The V/K pH-rate profiles exhibit pKas of reactant functional groups
prior to binding of the final reactant and of the other bound re-
actants, while V will exhibit pKas of bound reactant functional
groups. Thus, V1 will exhibit pKas of bound AASA and Glu, respec-
tively, and V1/KGlu will exhibit pKas of bound AASA and free Glu,
while V2 will exhibit pKas of bound Sacc and V2/KSacc will exhibit
pKas of free Sacc.
a
Once reactants are bound, the reaction would be expected to
proceed via Scheme 2. The
some enzyme residue. Then, as shown in Scheme 2, a nucleophilic
attack by the -amine of Glu on the aldehyde carbonyl of AASA
occurs as in I, with the carbonyl oxygen protonated by the -amine
a-amine of Glu is likely deprotonated by
a
a
of AASA, which remains hydrogen-bonded to the hydroxyl of the
newly formed carbinolamine. The carbinolamine hydroxyl accepts
a proton from the carbinolamine nitrogen assisted by the
a-
carboxylate of the Glu moiety to give the neutral carbinolamine, II
in Scheme 2. Water is eliminated to generate the imine, III in
Scheme 2, followed by reduction of the imine, IV in Scheme 2, to
give Sacc, V in Scheme 2.
As shown in Scheme 2, the
a-amine of Glu must be unproto-
nated to begin the reaction via a nucleophilic attack by the
a
-amine
3.5. Conclusions
on the aldehydic carbon of AASA in the direction of Sacc formation.
Thus, one of two scenarios must exist; either the enzyme selec-
Data are consistent with the acidebase mechanism proposed by
the QM/MM studies [8], i.e., direct catalysis by bound reactants as
suggested below.
tively binds Glu with a neutral
a-amine, or an enzyme group must
accept a proton from the -amine to start the reaction. The V1/KGlu
a
pH-rate profile exhibits a pKa of 5.6 on the acid side and a pKas of 7.9
and 8.5 on the basic side. The predominant forms of enzyme and
reactant under these conditions are the E-NADPH-AASA complex
and free Glu in solution. The group with a pKa of 5.7 likely reflects
1. The decrease in V/Et and V/KGluEt was ꢄ40-fold for all mutant
enzymes, suggesting that none are catalytically essential.
2. The V2/KSacc pH-rate profiles for all mutant enzymes decrease at
low and high pH, suggesting that acid and base catalytic resi-
dues are still present.
an enzyme group that accepts a proton from the
a-amine of Glu as
it binds to enzyme. This group is not D126, because the D126A