title: 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 p K (a) 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 Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).
authors.affiliation: Laboratory of Structural and Mechanistic Enzymology, School of Biological Sciences, Queen Mary, University of London, Mile End Road, UK.
authors.forename: Syeed
authors.initials: S
authors.lastname: Hussain
authors.forename: Surapong
authors.initials: S
authors.lastname: Pinitglang
authors.forename: Tamara S F
authors.initials: TS
authors.lastname: Bailey
authors.forename: James D
authors.initials: JD
authors.lastname: Reid
authors.forename: Michael A
authors.initials: MA
authors.lastname: Noble
authors.forename: Marina
authors.initials: M
authors.lastname: Resmini
authors.forename: Emrys W
authors.initials: EW
authors.lastname: Thomas
authors.forename: Richard B
authors.initials: RB
authors.lastname: Greaves
authors.forename: Chandra S
authors.initials: CS
authors.lastname: Verma
authors.forename: Keith
authors.initials: K
authors.lastname: Brocklehurst
mesh_terms: D000215:Acylation; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D010206:Papain / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D055672:Static Electricity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D004952:Esters; D013438:Sulfhydryl Compounds; D001224:Aspartic Acid; D003546:Cysteine Endopeptidases; C018745:actinidain; D010206:Papain