Effects of substituents on the rates of deacylation of substituted benzoyl papains. Role of a carboxylate residue in the catalytic mechanism

Abstract

The effect of ring substituents on the rates of deacylation of 8 meta- and para-substituted benzoyl papains was evaluated. The rate constants were found to depend upon a single ionizing group of pKa = 4.2--4.3, and to decrease by a factor of approximately 2.2 when measured in 94% D2O/H2O. The rates of deacylation are increased greatly by electron-withdrawing groups on the benzene ring. The Hammett rho value is 2.74 +/- 0.32. A plot of the rate constants for deacylation of the benzoyl papains against the corresponding constants for substituted benzoyl chymotrypsins generates a straight line of slope 1.0. This result suggests a very similar distribution of charge on the benzoyl moiety in the transition state for the two enzymes, which is interpreted in terms of the net charge of the transition state for the deacylation of nonspecific acyl papains being equal to--1 with the general base catalyzed assistance to the attack of water on the acyl enzyme being provided by the negatively charged Asp-158 rather than by the neutral Asn-175-His-159 hydrogen bond network. This result together with a survey of literature data suggests that the role of Asp-158 in papain catalysis has been underestimated. The evidence advanced to date in support of the proposition that an imidazolium-159-cysteine-25 thiolate ion pair exists in native papain is evaluated and considered to be insufficient to decide the issue.

Kinetic solvent isotope effects on the deacylation of specific acyl-papains. Proton inventory studies on the papain-catalysed hydrolyses of specific ester substrates: analysis of possible transition state structures

“…Thus it is apparent from the sigmoidal pH dependence of deacylation that the basic form of a group with PKa approx. 4 is required for catalytic activity (Whitaker & Bender, 1965;Williams & Whitaker, 1967;Lowe, 1976;Zannis & Kirsch, 1978). This type of pH-dependence has been interpreted, in common with the serine proteinases, in terms of general base catalysis (Fersht, 1977).…”

“…Such rotation is implied by the observation of acid catalysis of amide substrate hydrolysis (Lowe & Yuthavong, 1971), cryoenzymological studies (Angelides & Fink, 1978), active site thiol group modification studies (Brocklehurst & Malthouse, 1978;Brocklehurst et al, 1979a,b), and consideration of stereoelectronic constraints (Deslongschamps et al, 1975). Evidence for the involvement of Asp-158 in the catalytic mechanism has come from recent work on pH-rate profiles (Allen et al, 1978) and structure-reactivity correlations (Zannis & Kirsch, 1978). Consequently a charge relay system in papain is an intriguing possibility; it cannot be ruled out a priori.…”

Current problems in mechanistic studies of serine and cysteine proteinases

“…The validity of this assumption was questioned (Husain & Lowe, 1968) and was practically ruled out by the determination of the steric structure of the enzyme, which showed that the nearest carboxyl group, which belongs to aspartate-158, was at a distance of 0.75 nm from the sulphur atom of cysteine-25 (Drenth et al, 1970(Drenth et al, , 1976. Based on the magnitude of the Hammett p value for the rates of deacylation of substituted benzoyl-papains, Zannis & Kirsch (1978) claimed that it was most likely that the deacylation of nonspecific acyl-enzymes is catalysed by a carboxylate group as a general base. Referring to these data, Angelides & Fink (1979b) raised the possibility that deacylation of specific substrates could also be assisted by aspartate-158.…”

“…They concluded that the direct involvement of an uncharged histidine residue in the deacylation step in papain-catalysed reactions cannot be excluded. Johnson et al (1981 b) have also pointed out that the near-zero heat of ionization of the catalytic group of pKa = 4, another argument in favour of carboxylate participation (Zannis & Kirsch, 1978), is not inconsistent with the action of imidazole as a general base.…”

“…The two most important evidences were the spectroscopic detection of a mercaptide ion-like form of cysteine-25 (Polgar, 1974b), and the fluorometric detection of protonated imidazole (Sluyterman & de Graaf, 1970;Lowe & Whitworth, 1974;Sluyterman & Wijdenes, 1976). Zannis & Kirsch (1978) have not accepted the concept of ion-pair formation, and claimed that there was no proof for the participation of histidine-159 in fluorescence quenching. They suggested aspartate-158 as an alternative candidate.…”