Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase.

P.F Fitzpatrick,V Massey

doi:10.1016/s0021-9258(19)68169-x

Abstract

A mixture of cysteamine and glyoxylate, proposed by Hamilton et al. to form the physiological substrate of hog kidney D-amino acid oxidase (Hamilton, G. A., Buckthal, D. J., Mortensen, R. M., and Zerby, K. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers, it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamine and glyoxylate with a second order rate constant of 84 min-1 M-1 at 37 degrees C, pH 7.5. Steady state kinetic analyses reveal that thiazolidine-2-carboxylic acid is a better substrate at pH 8.5 than at pH 7.5. At both pH values, the catalytic turnover number is similar to that obtained with D-proline. D-Amino acid oxidase is rapidly reduced by thiazolidine-2-carboxylic acid to form a reduced enzyme-imino acid complex, as is typical with D-amino acid oxidase substrates. The product of oxidation was shown by NMR to be delta 2-thiazoline-2-carboxylic acid. Racemic thiazolidine-2-carboxylic acid is completely oxidized by the enzyme. The directly measured rate of isomerization of L-thiazolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by D-amino acid oxidase. Their identity over the range of temperature from 2-30 degrees C established that the apparent activity with the L-amino acid can be explained quantitatively by the rapid, prior isomerization to D-thiazolidine-2-carboxylic acid.

Highlights

A c d Sci U.S A. 76,2625-2629), was confirmed to act as a good substrate for the pure enzyme. As proposed by those workers,it was shown that the actual substrate is thiazolidine-2-carboxylic acid, formed from cysteamineand glyoxylate with a second order rate constant of 84 min” M” a t 37 “C, pH 7.5
SCHEME 1 zolidine-2-carboxylic acid to the D-isomer was compared to the rate of oxidation of the L-isomer by Damino acid oxidase
The assay followed the loss of thecysteamine thiol, since Scheme1predicts that this would be the final step in the reaction.A direct plot of the observed rate uersus the concentration of thevariedsubstrate, glyoxylate,gavea straight line with a zero intercept (Fig. 1).The second order rate constant calculated from these resultws as 84 min" M"

Summary

THEJOURNALOF BIOLOGZCCAHLEMISTRY

Thiazolidine-2-carboxylicAcid, an Adduct of Cysteamine and Glyoxylate, as a Substrate forD-Amino Acid Oxidase (Received for publication, March 9, 1981). One, cysteamine, when combinedwith glyoxylate, was active as a substrate for D-amino acid oxidase They proposed that the actual substratoexidized was thiazolidine-2-carboxylic acid,formed as in Scheme 1. With cyclic amino acids such as D-prOhe, the a-iminaocid is the final product; withnoncyclic amino acids the a-iminoacid enzyme flavin is reduced extremely rapidly by D-thiazolidine2-carboxylic acid but only slowly by the L-isomer. These results indicate that~-thiazolidine-2-carboxya~ciicd behaves as a normal substratefor the enzyme, withreaction properties very similar to those documented previously with D-proline (2, 5)

EXPERIMENTAL PROCEDURES

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DISCUSSION