Isolation and primary structure of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis hook.

The dried latex of the mountain papaya, Carica candamarcensis, was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-alpha-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCIa and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88-89% similarity with the three subvariants (G90/R212, E90/R212, E90/K212) of CCIa. 139-140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys25, His159, Asn175) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed.

Primary structure of CC-III, the glycosylated cysteine proteinase from the latex of Carica candamarcensis Hook.

The amino acid sequence of the cysteine proteinase CC-III from the latex of the subtropical species Carica candamarcensis Hook has been determined with the exception of seven residues (pos. 180-186). It was deduced from the sequence analysis of the whole chain and peptides obtained by tryptic, chymotryptic, peptic and thermolysinolytic hydrolysis. CC-III consists of 214 amino acid residues. Out of a total of eight cysteine residues, six are located at positions involved in the formation of the three disulfide bridges stabilizing the structure of papain related enzymes. CC-III from Carica candamarcensis is a glycoprotein with the carbohydrate moiety bound to asparagine at position 44. Out of 210 residues compared with the sequences of the four cysteine proteinases of Carica papaya L., CC-III shares 125 identical ones (59.5%) with papain, 142 (67.6%) with papaya proteinase IV, 146 (69.5%) with papaya proteinase III and 156 (74.3%) with chymopapain. All amino acid residues constituting the active site and subsite S2 in chymopapain are conserved in CC-III with the exception of the substitution Leu157--> Val in the latter. This fact as well as the highest degree of identity between CC-III and chymopapain point to a similar specificity of both enzymes and thus CC-III might be a suitable substitute for chymopapain as a chemonucleolytic agent.