Purification and properties of a mouse ascites tumor dipeptidase, a metalloenzyme.

A dipeptidase that hydrolyzes L-Ala-Gly and a wide spectrum of other L-ar-dipeptides has been purified 800-fold from the soluble fraction of Ehrlich-LettrB mouse actesis tmnor cells. The highest specific activity (micromoles of dipeptide hydrolyzed at 40” per min per mg of protein) achieved was 2,600 with Ala-Gly, the substrate with which purification was followed. With the best substrate, Ala-Ile, this specific activity was equivalent to a molecular activity (moles of dipeptide hydrolyzed at maximum velocity at 40” per min per mole of enzyme) of 2 X 106. Instability of the enzyme at this high activity prevented determination of homogeneity; a sample of molecular activity of 1 X lo6 (AlaGly specific activity, 1,360) was estimated to be 50% pure by acrylamide gel electrophoresis. Studies with metal chelators indicate that the dipeptidase is a metalloenzyme. For instance, o-phenanthroline completely inhibited the enzyme activity (50% at 0.1 mu), whereas m-phenanthroline had no effect. Although atomic absorption analyses showed a correlation of zinc content with enzyme activity in the final chromatographic step and a value of 0.9 f 0.1 g atom of zinc per mole of enzyme of specific activity 2,600 (Ala-Gly), it has not been confirmed that the dipeptidase is a zinc metalloenzyme. The enzyme was separated from leucine aminopeptidase, prolidase, and tripeptidase by Sephadex G-150 filtration and, by comparison with standard proteins, was shown to have a molecular weight of 85,000 f 5,000. The dipeptidase hydrolyzes only L-cr-dipeptides with a free amino and carboxyl group. Kinetic studies of 15 dipeptides have shown the K, values to vary between 0.4 and 22 mu and the relative V,,, values (Ala-Gly standard, V,,, = 1) to vary from 5.3 (Ala-lle) to 0.01 (Gly-Gly). Inhibition by high substrate concentrations (3 to 50 mu) was observed in cases where the Km was low. Peptides with small NH*terminal and bulky nonpolar COOH-terminal R groups were preferred. The hydrolyses of the relatively poor substrates, Pro-Gly and Gly-Gly, were activated by mu Mn2+ and Co2f, respectively, whereas that of Ala-Gly was inhibited by both these metals as well as by 10 other metals. No evidence was obtained that these hydrolyses were carried out by