Influence of modulated structural dynamics on the kinetics of α-chymotrypsin catalysis: Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Abstract

Although the chemical nature of the catalytic mechanism of the serine protease α-chymotrypsin (α-CT) is largely understood, the influence of the enzyme's structural dynamics on its catalysis remains uncertain. Here we investigate whether α-CT's structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation [Solá RJ & Griebenow K (2006) FEBS Lett580, 1685-1690] was used to generate a series of glycosylated α-CT conjugates with reduced structural dynamics, as determined from amide hydrogen/deuterium exchange kinetics (kHX). Determination of their catalytic behavior (KS, k2, and k3) for the hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) revealed decreased kinetics for the catalytic steps (k2 and k3) without affecting substrate binding (K S) at increasing glycosylation levels. Statistical correlation analysis between the catalytic (ΔG≠ki) and structurally dynamic (ΔGHX) parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the α-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing the Gaussian network model revealed structural insights into the relation between the protein's surface glycosylation, the resulting structural dynamic changes, and the influence of these on the enzyme's collective dynamics and catalytic residues. The experimental and theoretical results presented here not only provide fundamental insights concerning the influence of glycosylation on the protein biophysical properties but also support the hypothesis that for α-CT the global structural dynamics directly influence the kinetics of enzyme catalysis via mechanochemical coupling between domain motions and active site chemical groups. © 2006 The Authors.