Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives

Abstract

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.

Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding

“…The isolation procedures for most of the enzymes have been described previously: papain (Baines & Brocklehurst, 1979); actinidin (Brocklehurst et al, 1981b); papaya proteinase Ql (papaya peptidase A) (Baines & Brocklehurst, 1982;Brocklehurst & Salih, 1983); bovine spleen cathepsin B (Willenbrock & Brocklehurst, 1984); chymopapain A (Baines et al, 1986). Chymopapains B1-B3 were isolated from the dried latex of Carica papaya supplied by Powell & Scholefield, Liverpool, U.K., by ion-exchange chromatography as described for the enzymes of fresh non-fruit papaya latex by Brocklehurst et al (1985b).…”

Structural similarity of chymopapain forms as indicated by circular dichroism

“…On the other hand, very little structural information for the other two enzymes is available in the literature. The fraction known as chymopapain has been separated into several forms by cationexchange chromatography [4][5][6]; two main components, named chymopapains A and B, seem to be different in the reactivity of the thiol group at the active site [7]. However, Buttle & Barrett [4] have shown that the multiple forms of chymopapain are immunologically identical, suggesting that the chromatographic multiplicity of this proteinase is the result of post-translational modifications [8].…”