FORMATION OF AN INTERMEDIATE AND ITS RATE OF CONVERSION TO PYRUVATE DURING THE TRYPTOPHANASE-CATALYZED DEGRADATION OF S-O-NITROPHENYL-L-CYSTEINE

The tryptophanase-catalyzed degradation of S-o-nitrophenyl-l-cysteine (SOPC) to o-nitrothiophenol, pyruvate, and ammonia was examined by noting changes in SOPC directly at 330 nm or by coupling the appearance of pyruvate to the oxidation of NADH catalyzed by lactic dehydrogenase. A discrepancy between these two rates suggested the appearance of a solution intermediate which was subsequently trapped by reduction with NaB3H4. The resulting [3H]alanine was shown to be racemic by oxidative deamination with d-amino acid oxidase and l-glutamic dehydrogenase. When 200-400 µg of tryptophanase was used with 163-650 µg of lactic dehydrogenase in a coupled reaction mixture, the appearance of pyruvate followed first-order kinetics with the same first-order rate constant, k = 27.1 ± 1.08 min-1 at 30 °C. Therefore, the rate-limiting step under these conditions is the nonenzymatic conversion of an intermediate to pyruvate. On the basis of these data and the experiments of Vederas et al. (1978) [Vederas, J. C., Schleicher, E„ Tsai, M.-D.,&Floss, H. G. (1978) J. Biol. Chem. 253, 5350-5354] which showed that the β carbon of pyruvate was stereospecifically protonated during the tryptophanase catalyzed degradation of tryptophan, it is concluded that a-iminopropionate or the carbinolamine of pyruvate and ammoia together with o-nitrothiophenol are the immediate products of the tryptophanase-catalyzed degradation of SOPC. The intermediate undergoes a measurable rate of conversion to pyruvate and ammonia. © 1979, American Chemical Society. All rights reserved.