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).

The co-crystal structure of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) with a tripeptide fluoromethyl ketone (Z-VAE(OMe)-FMK)

“…Previous studies in papain indicated that the reactivity of the catalytic cysteine is due to formation of an ion-pair with the catalytic histidine, analogous to the activation of the catalytic serine in the eponymous serine proteases by histidine. 26 UCHL1 has been thought to be papain-like in mechanism of action, but our observation suggests that this may not necessarily be the case. The cysteine may be reactive enough without participation by the histidine, perhaps due to a lowered pK a .…”

Steady-State and Pre-Steady-State Kinetic Evaluation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CLpro Cysteine Protease: Development of an Ion-Pair Model for Catalysis

“…The evaluation of ester hydrolysis as a function of substrate concentration allowed for the estimation of the rate constants of the kinetic mechanism and is consistent with a double displacement (ping pong) mechanism. Unlike related enzymes (23,38,39), the burst rate of 3CL promediated ester hydrolysis was shown to be independent of substrate concentration. The 3CL pro burst rate observations can be accounted for if there was a high commitment to catalysis: the acylation rate (k 2 ) is much faster than the substrate dissociation rate (k -1 ).…”

“…Unlike the ester substrates, the amide substrates have a hydrogen bond donor in the scissile bond, which has been shown to have a significant role in catalysis for other proteases (23,(30)(31)(32)(33). Because of insufficient solubility and higher K m values, peptides 4 and 5 could not be saturated under pre-steady-state experimental conditions.…”

Modeling the effect of temperature and high hydrostatic pressure on the proteolytic activity of kiwi fruit juice

“…Actinidin has a wide substrate specificity, hydrolyzes most strongly the amide and ester bonds at the carboxyl side of a lysine residue and it is active at pH range 4-10 (Arcus, 1959;McDowall, 1970;Hussain et al, 2003;Morimoto et al, 2006). It is composed of 220 amino acid residues with molecular mass of 23.5 kDa (Carne and Moore, 1978;Baker, 1980;Watts and Brocklehurst, 2004).…”