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Abstract Crystalline tryptophanase from Escherichia coli catalyzes a variety of α,β elimination and β replacement reactions corresponding, respectively, to Equations i and ii. RCH2CHNH2COOH + H2O→CH3COCOOH + RH + NH3 (i) RCH2CHNH2COOH + indole→tryptophan + RH (ii) The initial rates of these reactions were followed spectrophotometrically by measuring pyruvate formation in the presence and absence of indole by use of a coupled system containing reduced diphosphopyridine nucleotide and lactate dehydrogenase. The affinity of tryptophanase for a given substrate is the same in Reaction i as in Reaction ii; consequently, association of the enzyme with indole, for which it shows high affinity (Km = 0.01 mm), does not result in any conformational change in the active site that is reflected in its affinity for its other substrates. Unlike indole, even high concentrations of hydrogen sulfide or methylmercaptan do not inhibit pyruvate formation in Reaction i and hence do not replace indole in β replacement reactions analogous to ii, although the corresponding amino acids, cysteine and S-methylcysteine, are excellent substrates for both reactions. These reactions are visualized as occurring by the mechanism outlined in Equations iii to v, where E represents tryptophanase. E + tryptophan (b)/⇌ E·amminoacrylate + indole (iii) E + RCH2CHNH2COOH ⇌ E·RCH2CHNH2COOH (b)/→ E·amminoacrylate + RH (iv) E·amminoacrylate + H2O → E + pyruvate + NH3 (v) Three kinetic cases can be distinguished: (a) the formation of pyruvate from tryptophan (Reactions iii + v), (b) the formation of pyruvate from other substrates (Reactions iv + v), and (c) the formation of tryptophan from other substrates plus indole (Reactions iv and iii in reverse). Rate equations derived for each of the three cases were tested graphically and were satisfied by kinetic data obtained with each of the five substrates (l-tryptophan, l-serine, l-cysteine, S-methyl-l-cysteine, and S-ethyl-l-cysteine) tested. In accordance with the proposed mechanism, an essentially identical value was obtained with all substrates for the kinetic parameter corresponding to the affinity constant of indole for the enzyme-aminoacrylate complex, the common intermediate in Reactions i and ii. This common intermediate decomposes to form tryptophanase, pyruvate, and ammonia at a rate approximately equal to that of dissociation of the enzyme-tryptophan complex. The maximal velocity of pyruvate formation from a given substrate in Reaction i is essentially identical with that of tryptophan synthesis from that same substrate in Reaction ii. Differences in the maximal velocity (Vmax) of Reactions i and ii are observed for different substrates, and these differences, considered in relation to the physical meaning of Vmax in terms of individual rate constants, provide strong kinetic evidence that the rate-determining step in each of the tryptophanase-catalyzed reactions lies in the removal of the β substituent from the substrate (Step b of Reactions iii and iv).
Morino et al. (Thu,) studied this question.