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On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes
Author(s) -
Kilshtain Alexandra Vardi,
Warshel Arieh
Publication year - 2009
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.22466
Subject(s) - chemistry , catalysis , catalytic cycle , mechanism (biology) , computational chemistry , enzyme catalysis , reaction mechanism , valence (chemistry) , chemical physics , thermodynamics , physics , organic chemistry , quantum mechanics
Zinc metalloenzymes play a major role in key biological processes and carboxypeptidase‐A (CPA) is a major prototype of such enzymes. The present work quantifies the energetics of the catalytic reaction of CPA and its mutants using the empirical valence bond (EVB) approach. The simulations allow us to quantify the origin of the catalytic power of this enzyme and to examine different mechanistic alternatives. The first step of the analysis used experimental information to determine the activation energy of each assumed mechanism of the reference reaction without the enzyme. The next step of the analysis involved EVB simulations of the reference reaction and then a calibration of the simulations by forcing them to reproduce the energetics of the reference reaction, in each assumed mechanism. The calibrated EVB was then used in systematic simulations of the catalytic reaction in the protein environment, without changing any parameter. The simulations reproduced the observed rate enhancement in two feasible general acid‐general base mechanisms (GAGB‐1 and GAGB‐2), although the calculations with the GAGB‐2 mechanism underestimated the catalytic effect in some treatments. We also reproduced the catalytic effect in the R127A mutant. The mutation calculations indicate that the GAGB‐2 mechanism is significantly less likely than the GAGB‐1 mechanism. It is also found, that the enzyme loses all its catalytic effect without the metal. This and earlier studies show that the catalytic effect of the metal is not some constant electrostatic effect, that can be assessed from gas phase studies, but a reflection of the dielectric effect of the specific environment. Proteins 2009. © 2009 Wiley‐Liss, Inc.