Inhibition and metal ion activation of pig kidney aminopeptidase P. Dependence on nature of substrate.
Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to homogeneity after its solubilisation from brush border membranes by phosphatidylinositol-specific phospholipase C. The effects of various activators and inhibitors of AP-P activity have been examined with a number of different substrates for the enzyme. The hydrolysis of bradykinin and ArgProPro is inhibited at Mn2+ concentrations above 10(-5) M, whereas the hydrolysis of other substrates (GlyProHyp, beta-casomorphin, substance P) is substantially activated, with 4-10 mM Mn2+ being optimal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of bradykinin. A number of inhibitors of angiotensin converting enzyme (ACE; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyProHyp, have no effect on the hydrolysis of bradykinin except in the presence of Mn2+. Differences were also observed in the degree of inhibition of GlyProHyp and bradykinin hydrolysis by EDTA and their reactivation by divalent cations. The hydrolysis of GlyProHyp follows Michaelis-Menten kinetics with a Km value of 2.7 mM. Bradykinin inhibits GlyProHyp hydrolysis with an I50 of 1.4 microM. The hydrolysis of bradykinin by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this substrate. These results indicate that substrates for AP-P can be divided into 2 groups based on their responses to inhibitors and cation activators.
Lloyd
G S
GS
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Hryszko
J
J
Hooper
N M
NM
Turner
A J
AJ
D000626:Aminopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D002712:Chlorides / Q000494:pharmacology*; D003035:Cobalt / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D017895:Manganese Compounds / Q000494:pharmacology*; D008871:Microvilli / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
D000806:Angiotensin-Converting Enzyme Inhibitors; D002712:Chlorides; D004793:Enzyme Reactivators; D017895:Manganese Compounds; D003035:Cobalt; D004492:Edetic Acid; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; C025340:manganese chloride