Biomedical Engineering Reference
In-Depth Information
Interpretation of experimental data on the proton inventory technique is difficult because
of the products of multiple effects, but also because of the uncertain of influence of
solvent isotope composition on pKa of enzyme functional groups and on electrostatic
interactions between charges. Changes of the vibrational properties of water molecules
inside enzyme globules and the complexity of multistep mechanisms can also complicate
an unequivocal interpretation. To avoid these difficulties, high pressure was proposed as
a perturbant (Northrop and Cho, 2000). It is suggested that high pressure simply changes
distribution within preexisting equilibrium, and pressure effects can separate multiple
isotopes effects from each other. When applied in conjunction with substrate isotope
effects, a primary kinetic assignment can distinguish between concerted and stepwise
chemical mechanisms of enzymatic catalysis.
Effects of high pressure on the kinetic parameter V/K are given by Eq. 1.22:
where p is the pressure, is the product ratio of forward and reverse enzymatic
constant, is the equilibrium constant for transition between two non-active and
active enzyme conformations, is the forward commitment (Eq. 1.18); and
is the difference in volume at the formation of the enzyme-substrate complex,
the transition state, and the conformational transition, respectively. Eq. 1.22
predicts at least biphasic dependence of (V/K)p on pressure originating from the
changes of the volume at limiting chemical steps and at the enzyme conformational
transition.
This method was used for investigating the mechanism of oxidation of benzyl
alcohol by yeast alcohol dehydrogenase (YADH). In this reaction an intrinsic tritium
effect is fully expressed in V/K. At pressure up to approximately 1.5 kbar, changes in
(V/K)p or the substrate binding were directly proportional to pressure, probably because
of the increase of the rate constant for hybrid transfer due to its negative activation
volume. The subsequent decrease in binding at higher pressure was shown to be due to a
positive volume change in conformational transition of complex. Such a
transition involves considerable solvent reorganization and, therefore, leads to a solvent
isotope effect. The experimental ratio first increased as pressure
increased up to 1.5 kbar and, then decreased. Thus, the pressure effect on the reaction is
stronger in than that in The extremely small sum of commitments
shows that the hydrid transfer is the reaction limiting step.
1.2..2. TRANSITION STATE ANALOGS METHODS
In 1946 Pauling introduced idea that lowering of the activation energy in enzyme
catalysis stems from the enzyme's affinity for the transition state exceeding it's affinity
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