Arginine racemase of Pseudomonas graveolens. II. Racemization and transamination of ornithine catalyzed by arginine racemase.

Arginine racemase from Pseudomonas graveolens is inactivated by preliminary incubation with Lor D-ornithine, 6-Nacetyl-L-ornithine, and L-2,4-diaminobutyrate at pH 10.0. Loss of the enzyme activity is associated with formation of a new spectrum with an absorption maximum in the region of 320 rnp and disappearance of a peak at 420 mp. The inactivation is prevented, and the inactivated enzyme is reactivated by addition of pyruvate or oxaloacetate. Addition of pyridoxal 5’-phosphate has substantially no effect. Exhaustive dialysis of the enzyme treated with ornithine leads to loss of the absorption at about 320 rnp to yield apoenzyme. The apoenzyme is reactivated by adding either pyridoxal 5’-phosphate alone or pyridoxamine St-phosphate plus pyruvate. Inactivation appears to be due to formation of bound pyridoxamine 5’-phosphate from bound pyridoxal 5’-phosphate by transamination with ornithine, which is converted into A’-pyrroline-2-carboxylic acid, the intramolecularly dehydrated form of cY-keto-6-aminovaleric acid. Pyridoxamine S’-phosphate formed is tightly bound to the protein moiety and dissociates from the enzyme only after long dialysis. The enzyme catalyzes transamination between Lor Dornithine (or certain other amino acids) and pyruvate (or certain other cY-keto acids). There is correlation between the amino acids that cause inactivation and those that transaminate, and between the cY-keto acids that reactivate the inactivated enzyme and those that transaminate. The rate of transamination is very low as compared to that of racemization. The enzyme exhibits optimal activity at about pH 11 for transamination. The racemase is only slightly inactivated by incubation with ornithine in the lower pH region in which the rate of transamination is low.