tailieunhanh - Báo cáo khoa học: Influence of modulated structural dynamics on the kinetics of a-chymotrypsin catalysis Insights through chemical glycosylation, molecular dynamics and domain motion analysis

Although the chemical nature of the catalytic mechanism of the serine pro-tease a-chymotrypsin (a-CT) is largely understood, the influence of the enzyme’s structural dynamics on its catalysis remains uncertain. Here we investigate whether a-CT’s structural dynamics directly influence the kinet-ics of enzyme catalysis. | ỊFEBS Journal Influence of modulated structural dynamics on the kinetics of a-chymotrypsin catalysis Insights through chemical glycosylation molecular dynamics and domain motion analysis Ricardo J. Sola and Kai Griebenow Laboratory for Applied Biochemistry and Biotechnology Department of Chemistry University of Puerto Rico Rio Piedras Campus San Juan PR USA Keywords a-chymotrypsin enzyme catalysis glycosylation molecular dynamics serine protease Correspondence K. Griebenow Department of Chemistry University of Puerto Rico Rio Piedras Campus Facundo Bueso Bldg Laboratory-215 San Juan 23346 PR 00931-3346 USA Fax 1 787 756 7717 Tel 1 787 764 0000 E-mail griebeno@ Received 5 July 2006 revised 26 September 2006 accepted 4 October 2006 doi Although the chemical nature of the catalytic mechanism of the serine protease a-chymotrypsin a-CT is largely understood the influence of the enzyme s structural dynamics on its catalysis remains uncertain. Here we investigate whether a-CT s structural dynamics directly influence the kinetics of enzyme catalysis. Chemical glycosylation Sola RJ Griebenow K 2006 FEBS Lett 580 1685-1690 was used to generate a series of glycosylated a-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 KS at increasing glycosylation levels. Statistical correlation analysis between the catalytic AG k and structurally dynamic DGHX parameters determined revealed that the enzyme acylation and deacylation steps are directly influenced by the changes in protein structural dynamics. Molecular modelling of the a-CT glycoconjugates coupled with molecular dynamics simulations and domain motion analysis employing .