Porcine liver aminopeptidase. Further characterization of its sulfhydryl groups.
Porcine liver aminopeptidase was inactivated by various sulfhydryl-reactive reagents, whose inactivation rates were in the order: p-chloromercuribenzoate(PCMB) greater than HgCl2 greater than 2,2'-dithiodipyridine greater than 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB). The processes of inactivation by these reagents did not follow pseudo-first-order kinetics, and prolonged incubation did not alter the level of maximum inactivation. The substrates provided no protection against the inactivation by DTNB, and the numbers of sulfhydryl groups titrated with the reagent were not influenced by the presence or absence of puromycin (a competitive inhibitor). The modification of sulfhydryl groups caused a slight increase in the Km value for the enzyme and a significant decrease of the Vmax value. There are two ionizable groups (pKe, 6.2; 7.8 and pKes, 6.0; 7.8) in the catalytic action of the enzyme. From the pKi vs. pH profile of inhibition with PCMB, the pK value of 7.8 does not correspond to the ionization of a sulfhydryl group. The thiol-modified enzyme was activated by cobalt ion, as was the native enzyme (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101). But in contrast with the native enzyme, the thiol-modified enzyme was activated about 2.5-fold and the maximum activation remained almost constant during prolonged incubation with cobalt ion. These results suggest that the sulfhydryl groups of the enzyme are located apart from the binding site of cobalt ion and do not participate directly in the catalytic process.
D000626:Aminopeptidases / Q000037:antagonists & inhibitors; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003035:Cobalt / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013552:Swine; D013997:Time Factors
D013438:Sulfhydryl Compounds; D003035:Cobalt; D004228:Dithionitrobenzoic Acid; D000626:Aminopeptidases