A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

Citation
Ym. Ayala et E. Di Cera, A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases, PROTEIN SCI, 9(8), 2000, pp. 1589-1593
Citations number
19
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
9
Issue
8
Year of publication
2000
Pages
1589 - 1593
Database
ISI
SICI code
0961-8368(200008)9:8<1589:ASMFTD>2.0.ZU;2-W
Abstract
A simple method is presented for the determination of individual rate const ants for substrate hydrolysis by serine proteases and other enzymes with si milar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrat e diffusion into the arrive site (k(1)), substrate dissociation (k(-1)), ac ylation (k(2)), and deacylation (k(3)) in the accepted mechanism of substra te hydrolysis by serine proteases are derived from the temperature dependen ce of the Michaelis-Menton parameters k(cat)/K-m and k(cat). The method als o yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the cataly tic mechanism are affected by Nai binding and site-directed mutations of th e important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determ inants of protease function.