Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.
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.
Reid
James D
JD
Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Hussain
Syeed
S
Bailey
Tamara S F
TS
Sonkaria
Sanjiv
S
Sreedharan
Suneal K
SK
Thomas
Emrys W
EW
Resmini
Marina
M
Brocklehurst
Keith
K
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*
D000097:Acetonitriles; C485101:N-acetyl-phenylalanyl-glycine methyl thionester; D009842:Oligopeptides; D012997:Solvents; D003546:Cysteine Endopeptidases; C018745:actinidain; D010206:Papain; C032159:acetonitrile