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We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.

in the second 0.870. In accordance with Student's criterion a probability of the coincidence of these mean values is below 0.01. Moreover in the correlations employing , the reaction sets of different authors under equivalent or very like conditions lead to the same correlation parameters. For example, sets 2, 4, and 6, respectively, yield p 1.07, 1.09, and 1.05 and pK°= 0.87, 0.98, and 1.00. Sets 3 and 5 give p 1.90 and 1.66, and pK°= 5.93 and 6.17. Differences in these values do not exceed the mean deviations. The correlation with Charton's method gives substantially different parameters. The sets 2, 4, and 6 give h = 2.31, 0.23, and 3.02, a = -6.83, -4.78, and -3.78, ß = -3.02, -4.05, and -0.62, respectively. The sets 3 and 5 give h = 7.99 and 7.26, a = -13.30 and -12.18, ß = 0.44 and -4,80. There is obviously no physical sense in these differences. Thus the Charton correlations, although giving satisfactory results, may lead to serious errors when analyzing the substituent effects on the properties of organophosphorus compounds. This danger may be avoided by using the constants.The correlation of with and using eq 7gives approximately linear dependence with the de-

Abstract. As a result of alkyl substitution in both aromatic ring and side chain, the rate constant for acid-catalyzed lactonization of hydrocoumaric acid is increased by factors as high as 1011 and, in comparison with the bimolecular esterification of phenol and acetic acid, by almost 1016. In the most favorable case studied, the half-life of the phenolic aeid (imidazole buffer, pH 7, 30'C) is 6 sec, with 90% of the total rate being due to catalysis by the buffer species. The effect is attributed to a unique interlocking of methyl groups, which produces a severe conformational restriction of the side chain and increases greatly the population of the most productive conformer. This phenomenon is presented as a model for the conformational restraint imposed by an enzyme on its substrate. The magnitude of the effect suggests that the contribution of substrate "freezing" to total rate enhancement by an enzyme can be considerably greater than previously supposed and may in fact be sufficient to complete the justification for enzyme catalysis.The enormous rate advantage of an enzyme-promoted reaction over its nonenzymatic counterpart may, under comparable conditions, extend beyond 1015. The enzymic process is considered to benefit from the synergistic combination of several rate-enhancing features, including proximity, distortion, general acidbase catalysis, and conformational restriction.1'2 Despite the use of liberal estimates for the contributions of these kinetic factors, a sizeable portion of the total rate enhancement remains unexplained. In this report our concern is with the last mentioned of the contributing factors, conformational restriction.The combined effects of multi-point attachment and close fit of a substrate to an enzyme surface serve to produce a unique orientation of the substrate with respect to catalytic and reactive sites on the enzyme and to "freeze" the substrate into a unique conformation with respect to its internal nonbonded interactions.3 Such restriction in rotational freedom of a substrate by the enzyme is comparable to any chemical alteration of an organic compound which serves to narrow the distribution of conformational populations, ideally by eliminating nonproductive isomers. Stereopopulation control is a convenient general term for any such manipulation. Although the value of this factor to an enzyme has been rated at 103-104, such estimates are based on meager quantitative data and on limited types of model systems.Several years ago, Thanassi and Cohen4 proposed a cyclic scheme for mitochondrial oxidative phosphorylation that required the esterification of ubihydro-1143

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