title: Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.
abstract: The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes.
authors.affiliation: Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
authors.forename: James D
authors.initials: JD
authors.lastname: Reid
authors.forename: Syeed
authors.initials: S
authors.lastname: Hussain
authors.forename: Tamara S F
authors.initials: TS
authors.lastname: Bailey
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Sonkaria
authors.forename: Suneal K
authors.initials: SK
authors.lastname: Sreedharan
authors.forename: Emrys W
authors.initials: EW
authors.lastname: Thomas
authors.forename: Marina
authors.initials: M
authors.lastname: Resmini
authors.forename: Keith
authors.initials: K
authors.lastname: Brocklehurst
mesh_terms: D000097:Acetonitriles / Q000737:chemistry*; D000215:Acylation; D002384:Catalysis; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D012997:Solvents / Q000737:chemistry*
chemical_list: D000097:Acetonitriles; C485101:N-acetyl-phenylalanyl-glycine methyl thionester; D009842:Oligopeptides; D012997:Solvents; D003546:Cysteine Endopeptidases; C018745:actinidain; D010206:Papain; C032159:acetonitrile