Amino acid sequence involving the reactive thiol group of ficin

Multiple Molecular Forms of Ficus glabrata Ficin. Their Separation and Relative Physical, Chemical, and Enzymatic Properties

“…The realization that Ficus glabrata ficin is heterogeneous and the difficulty of isolating, in pure form, the enzyme components have slowed work on the enzyme. Limited data are avXailable on the specificity (9,13), the kinetic parameters (1,7,8) and the amino acid sequence around the essential sulfhydryl group of ficin (28). However, these data were obtained with preparation-s containing all the multiple molecular forms.…”

The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residues in ficin

“…Ficin is irreversibly inhibited through a reactive cysteine residue by iodoacetamide (Hollaway, Mathias & Rabin, 1964), by iodoacetate (Wong & Liener, 1964), by the chloromethyl ketones derived from N-toluene-p-sulphonyl-L-lysine and N-toluene-p-sulphonyl-L-phenylalanine (Stein & Liener, 1967), by N-methylmaleimide (Hollaway et al 1964) and by DDPM* (Metrione, Johnston & Seng, 1967). Using iodo[14C]acetate-inhibited ficin Wong & Liener (1964) isolated a radioactive peptide, after enzymic digestion, that showed a high degree of homology with the amino acid sequence around the active-site cysteine of papain.…”

“…it is known that ficin contains two thiol groups, one that is reactive in the active enzyme and one that is only titratable withp-chloromercuribenzoate (Liener, 1961) or alkylated with iodoacetate (Englund, King, Craig & Walti, 1968) after the tertiary structure of the enzyme has been disrupted. Using DDPM to label ficin, Metrione et al (1967) isolated a coloured peptide, after peptic digestion of the DDPS-ficin, that was quite different in amino acid composition and amino acid sequence from that obtained by Wong & Liener (1964) and showed no homology with the amino acid sequence around the active-site cysteine of papain. Two possible explanations were offered by Metrione et al (1967).…”

“…Digestion of the radioactive peptide 1 with leucine aminopeptidase did not release glutamine. Since glutamine was expected from the work of Wong & Liener (1964) to be the N-terminal residue ofthe cysteine chain, it appeared probable that radioactive peptide 1 contained a rearranged or pyroglutamic acid residue at the Nterminus of the cysteine chain. Advantage was taken of this observation to determine the sequence of the cysteine-containing and histidine-containing chains in the radioactive peptides.…”

“…After nine cycles, no N-terminal residue was found, but after the tenth and eleventh cycles alanine and valine were the predominant N-terminal residues. As it was possible to assign the cysteine and histidine residues to the respective chains from the known sequence around the activesite cysteine residue (Wong & Liener, 1964), it only remained therefore to assign the C-terminal tryptophan and leucine to their respective chains. The radioactive peptide 2 was oxidized with performic acid and the ester bond formed from the crosslinking ketone was cleaved with hydroxylamine.…”