SPECIFICITY AND KINETICS OF THE MILK-CLOTTING ENZYME FROM CARDOON (CYNARA-CARDUNCULUS L) TOWARD BOVINE KAPPA-CASEIN

The action of Cynara cardunculus L. protease on whole bovine kappa-cas ein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3 % trichloroacetic acid (TCA)-soluble fraction of the kappa-casein diges tion mixture showed three peptide peaks, which were identified by amin o acid analysis and N-terminal analysis as the 106-169 fragment [casei nomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by s pectrofluorometry, using fluorescein isothiocyanate-labeled kappa-case in (FTC-kappa-casein). The values obtained for k(cat), k(m), and k(cat )/k(m) were 1.04 s-1, 0.16 muM, and 6.5 muM-1 s-1, respectively. The p roteolytic coefficient is of the same order of magnitude as those obta ined for other milk-clotting enzymes, but the k(m) is significantly lo wer, which reflects the higher affinity of Cynara protease to kappa-ca sein.