Selective cleavage of glycyl bonds by papaya proteinase IV

Abstract

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO, is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in p-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.

Inhibition of cysteine proteinases by a protein inhibitor from potato

“…On the other hand, the cysteine-dependent exopeptidase, dipeptidyl peptidase I, which is well inhibited by several cystatin-type inhibitors [18], was not detectably inhibited by PCPI. Also, papaya proteinase IV, perhaps the most specific known member of the papain superfamily [19] was unaffected by PCPI 8.3, as it is by the cystatins [20].…”

Factors effecting the thermostability of cysteine proteinases from Carica papaya

“…The proteinase identity was also determined by substrate specificity using Bz-Arg-NH-Ph, tBOC-Ala-Ala-Gly-NH-Ph, CBz-Phe-Val-Arg-NH-Ph (Bz, benzoyl; NH-Ph, p-nitroanilide; tBoc, N-tert-butoxycarbonyl; CBz, carbobenzoxy) and the differential response of the enzymes to chicken cystatin 1990b).…”

Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly9‐Ala10 bond

“…A similar procedure was used for the purification of papain from Boehringer (Mannheim). Papaya proteinase IV (PPIV) activity was assayed in 0.1 M phosphate buffer pH 6.8, 2 mM EDTA, 0.015% Brij 35, using the substrate Boc-Ala-Ala-Gly-NHPhNO2, and discriminated from papain activity by its inhibition with E-64 and lack of inhibition with chicken cystatin [11,12]. Active papain concentration was determined by titration with E-64 [13].…”