Conversion of the Active‐Site Cysteine Residue of Papain into a Dehydro‐serine, a Serine and a Glycine Residue

Abstract

Photolysis of papain which had been inhibited with 2-bromo-2',4'-dimethoxyacetophenone regenerated papain, but also formed [ A Ser2j ]papain (i.e. papain in which the active-site cysteine Kinetic specificity or the manifestation of enzyme specificity in the maximum velocity is a frequently encountered phenomenon in enzymology [I]. It is not explicable in terms of a simple structural complementarity between enzyme and substrate [2] and several alternative concepts have been suggested [3 -61. In order to gain a deeper understanding of the causes of kinetic specificity it is necessary to have detailed structural information of the enzyme-substrate complex.X-ray crystallography is at the present time the only method capable of providing sufficiently detailed structural information to determine the cause of kinetic specificity. Low-temperature X-ray crystallography is a promising approach for the study of enzyme-substrate complexes [7,8], but for the proteolytic enzyme, papain, where suitable crystals for highresolution X-ray crystallography were obtained only from methanol/water mixtures [9], a different approach seemed necessary.It was considered that if the active-site thiol group of Cys-25 in papain could be converted to a hydroxyl group, the modified enzyme (henceforward referred to as [ S e~~~I p a p a i n ) would retain its binding properties for peptides, but would be incapable of hydrolysing them. The chemical modification envisaged had the desirable feature of slightly reducing the steric requirement of the essential thiol group and therefore would not lead to steric exclusion of the substrate. Moreover the replacement of a thiol group by a hydroxyl is electronically the closest that could be envisaged and had the further desirable feature of leading to a natural amino acid, a process which has been referred to as 'chemical mutation' [lo]. It was expected that such a modification would not significantly perturb the tertiary structure of the enzyme. The conversion of the active-site serine residue of subtilisin into a cysteine residue was achieved [lo-121 without perturbation of the tertiary structure of the enzyme (J. Kraut, private communication). The conversion of a cysteine residue to a serine residue in a protein molecule has not hitherto been reported. The chemical strategy envisaged for this transformation is outlined in Scheme 1. a-Bromoketones selectively alkylate the active-site cysteine residue of papain. By using a phenacyl bromide with an absorption band at longer wavelength than that of the enzyme, photolysis should lead to Norrish type II cleavage 1131, provided that the protein would allow the required conformation to be adopted by the alkylated cysteine residue. Thiols have been converted into thioaldehydes by this method in non-hydroxylic solvents [13 -151, but it was anticipated that steric restriction arising from the protein structure [9] would

Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte

“…Importantly, gene disruption studies in P. falciparum have shown that only SERA5 (“Ser”-type) and the much less abundant SERA6 (“Cys”-type: P. falciparum 3D7 PlasmoDB ID PF3D7_0207500) are essential in asexual blood stages (12, 20). Experimental Cys to Ser substitution at the active site of a number of cysteine proteases, including papain (21), streptopain (22), and cathepsin L (23), abolishes or profoundly reduces the proteolytic activity of those enzymes, so the absence of a catalytic Cys in SERA5 has led to suggestions that it and the other Ser-type SERAs are unlikely to be proteases. In support of this, only very limited protease activity was found associated with a recombinant form of the SERA5 papain-like domain (24), and x-ray crystallographic studies of the same protein have revealed aberrant structural features that cast doubt on its capacity to interact in a canonical manner with polypeptide substrates (25).…”

Active site residues in m‐calpain: identification by site‐directed mutagenesis

“…Conversion of the papain active site cysteine residue to serine (as with the calpain C105S mutant) caused loss of activity, which was attributed to a difference in length between a C-O and C-S bond [15,16]. This may not however be the only factor involved.…”

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“…Chalker et al [37] carried out chemical modification of cystein molecule using keratinous protein lately in 2009. Whereas, in 1978, Clark and Lowe [38] had already worked on conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine Residue. They used bromoacetophenone derivatives to alkylate cysteine of keratin and converted into formyl glycine photochemically.…”