Variation in the pH-dependent pre-steady-state and steady-state kinetic characteristics of cysteine-proteinase mechanism: evidence for electrostatic modulation of catalytic-site function by the neighbouring carboxylate anion

Abstract

The acylation and deacylation stages of the hydrolysis of N-acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential pKa values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp161 is the source of a key electrostatic modulator (pKa 5.0±0.1) in actinidin, analogous to Asp158 in papain, whose influence is not detected kinetically; it is always in the ‘on’ state because of its low pKa value (2.8±0.06).