title: Enzymatic reaction of silent substrates: kinetic theory and application to the serine protease chymotrypsin.
abstract: Investigating the selectivity that an enzyme expresses toward its substrates can be technically challenging if reaction of these substrates is not accompanied by a conveniently monitored change in some physicochemical property. In this paper, we describe a simple method for determining steady-state kinetic parameters for enzymatic turnover of such "silent" substrates. According to this method, silent substrate S is allowed to compete for enzymic reaction with signal-generating substrate S*, whose conversion to product can be conveniently monitored. Full reaction progress curves are collected under conditions of [S*](o) << K(m)* and [S](o) >or= 3K(m). Progress curves collected under these conditions are characterized by an initial lag phase of duration tau that is followed by the pseudo-first-order reaction of S. Steady-state kinetic parameters for the silent substrate can be obtained by one of two methods. One method combines least-squares fitting with numerical integration of appropriate rate equations to analyze the progress curves, while the other method relies on direct graphical analysis in which K(m) is the value of [S](o) that reduces the control velocity by a factor of 2 and V(max) is shown to simply equal the ratio [S](o)/tau. We use these methods to analyze the alpha-chymotrypsin-catalyzed hydrolysis of silent substrate Suc-Ala-Phe-AlaNH(2) with signal generator Suc-Ala-Phe-pNA. From the curve-fitting method, k(c) = 0.9 +/- 0.2 s(-1) and K(m) = 0.4 +/- 0.1 mM, while by direct graphical analysis, k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.51 +/- 0.03 mM. As validation of this new method, we show agreement of these values with those determined independently by HPLC analysis of the hydrolysis of Suc-Ala-Phe-AlaNH(2) by alpha-CT, where k(c) = 1.1 +/- 0.1 s(-1) and K(m) = 0.5 +/- 0.1 mM.
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139, USA.
authors.forename: April
authors.initials: A
authors.lastname: Case
authors.forename: W Phillip
authors.initials: WP
authors.lastname: Huskey
authors.forename: Ross L
authors.initials: RL
authors.lastname: Stein
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; C473992:succinimidyl-alanyl-phenylalanyl-4-nitroaniline; C473993:succinimidyl-alanyl-phenylalanyl-alanine; D002918:Chymotrypsin