Rat liver arginase: kinetic mechanism, alternate substrates, and inhibitors.
The activity of guanidino compounds as alternate substrates for rat liver arginase is critically dependent on the length of the amino acid side chain and the substituents about C-alpha. In addition to L-arginine, the enzyme catalyzes the hydrolysis of L-argininamide, L-canavanine, L-homoarginine, L-argininic acid, and agmatine. The kcat values for these substrates are 15- to 5000-fold slower than the kcat for L-arginine. Guanidobutyrate, D-arginine, and NG-methyl-L-arginine are not substrates. Competitive inhibition by the products L-ornithine and urea indicates a rapid-equilibrium random mechanism for the enzyme. Despite the requirement for added divalent cations in the activation of the enzyme, metal chelators such as EDTA and citrate do not inhibit the enzyme. These results suggest that the metal site is not readily accessible to solvent. Multiple inhibition experiments with the noncompetitive inhibitor borate demonstrate that borate and urea bind in a mutually exclusive manner, while L-ornithine and borate can bind simultaneously to the enzyme. Borate inhibition is proposed to arise from chelation of Mn(II) in the binuclear Mn(II) center, thus displacing a metal-bound water molecule that is responsible for nucleophilic attack on the guanidium carbon.
Reczkowski
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Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Ash
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D000376:Agmatine / Q000378:metabolism; D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives; D001881:Borates / Q000494:pharmacology; D002172:Canavanine / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D006146:Guanidines / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008667:Metalloproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008956:Models, Chemical; D051381:Rats; D013379:Substrate Specificity
D001881:Borates; D002614:Chelating Agents; D006146:Guanidines; D008667:Metalloproteins; D002172:Canavanine; D000376:Agmatine; D001120:Arginine; D001119:Arginase