An improved colorimetric assay for carbamyl oxamate, which allows the precise measurement of the activity of oxamic transcarbamylase, has been developed. Activity is maximum over the pH range from 8.3 to 8.7. A cation requirement is satisfied by 2.5 X 10-3 M Mg++ or MnII. The equilibrium constant for the phosphorolysis of carbamyl oxamic acid is 1.6, corresponding to a negative free energy change of-285 cal per mole. The conversion of carbamyl oxamate to oxamate by Streptococcus allantoicus is a unique phosphorolytic reaction catalyzed by oxamic transcarbamylase (Valentine and Wolfe, 1960a; Bojanowski et al., 1962
1965.-A new spectrophotometric method for the determination of glyoxylate is described. The technique is based on measurement of the initial rate of formation of glyoxylic acid phenylhydrazone in neutral solution. Its advantages include rapidity and convenience, suitability for use with mixtures containing acid-labile substrates, and elimination of possibly inhibitory reagents from the enzyme incubation mixture. Ureidoglycolate synthetase, which cleaves ureidoglycolate to glyoxylate and urea, was purified from crude extracts of Streptococcus allantoicus grown on allantoin-containing medium. The purification procedures include treatment with MnCl2, fractionation on calcium phosphate gel, fractional precipitation with ammonium sulfate, and column chromatography on diethylaminoethyl cellulose. The final enzyme preparation was purified 77-fold and contained 35% of the total activity of the extract. In previous communications, we have proposed a scheme for the fermentation of allantoin by Streptococcus allantoicus (Valentine et al., 1962; Valentine and Wolfe, 1961; Gaudy et al., 1962). The conversion of allantoic acid to glyoxylate and urea in other organisms has been attributed to the action of a single enzyme, allantoicase (Laskowski, 1951; DiCarlo, Schultz, and Kent, 1953; Campbell, 1954). Present evidence indicates that in S. allantoicus at least two enzymes are involved in this cleavage; allantoic acid is hydrolyzed to urea and ureidoglycolate, the latter being cleaved to glyoxylate and urea by the enzyme ureidoglycolate synthetase. [In this communication the name, ureidoglycolate, is used in place of the former name, glyoxylurea, and ureidoglycolate synthetase is used in place of glyoxylurease (see Discussion).] NaOOC-CHOH-NH-CO-NH2 Ureidoglycolate ± CHO-COONa + NH2-CO-NH2 Glyoxylate Urea
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