Abstract
Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.
Highlights
Various metalloenzymes act in fundamental biological processes found in nature
Earlier studies that were conducted on glycerol dehydrogenase from Klebsiella pneumonia [23] and from other microorganisms [24,25] reported the Glycerol dehydrogenase (GDH) follow an ordered BiBi sequential mechanism
A simple, quick and valid strategy based on kinetic and thermodynamic analysis for understanding the significant mechanistic changes induced by metal ion substitution was proposed
Summary
Various metalloenzymes act in fundamental biological processes found in nature. The metal ion of most metalloenzymes participates in the catalytic process involved in the function of the polarization of chemical bonds, nucleophile activation, and substrate or product coordination [1]. Substitution of metal ions in metalloenzymes is a mild and effective modification that is used in structure-function relationship studies [2]. Metal ion substitutions have been reported to change the catalytic activity [3], substrate specificity [4], and stability [5,6] of metalloenzymes and have stimulated much research interest. The mechanism of metal ion substitution has been mainly studied by molecular simulation and the analysis of substituted enzyme crystals. D’Antonio investigated the structure of cobalt-reconstituted human arginase I, revealing the change of the catalytic mechanism upon metal ion substitution [8]. The molecular simulation of the enzyme structure and the generation of metal ion substituted enzyme crystals are lengthy and expensive processes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
