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The Relation of Spectral Changes and Tritium Exchange Reactions to the Mechanism of Tryptophanase-catalyzed Reactions

From the ¹ From the Department of Biochemistry, University of California, Berkeley, California 94720

Holotryptophanase in the presence of K⁺ undergoes a pH-dependent change (pK 7.2) from a protonated, enzymatically inactive form (EH), _max 420 mµ, to a deprotonated, active form (E), _max 337 mµ. Only the inactive form appears when K⁺, which is required for activity, is replaced by Na⁺ or imidazole. Both forms of the enzyme are completely inactivated by borohydride reduction, indicating that each contains an azomethine link between coenzyme and apoenzyme. -Pyridoxyllysine was isolated from the hydrolyzed reduced enzyme in amounts corresponding to the 4 moles of pyridoxal-P that were found by spectrophotometric titration to combine with the native apoenzyme (mol wt 220,000). Interaction with its substrates (or quasisubstrates) to form an ES complex partially protects tryptophanase against inactivation by borohydride reduction, indicating that such interaction replaces the azomethine link from coenzyme to protein with a similar linkage from coenzyme to substrate. On addition of substrates or of competitive inhibitors such as l-alanine, tryptophanase exhibits an absorption maximum near 500 mµ, which disappears as the substrates are decomposed, but remains unchanged in the presence of the inhibitors. Exchange experiments in the presence of D₂O or T₂O show that the hydrogen of l-alanine is labilized under these conditions. On these and other grounds, the 500 mµ band is ascribed to a quinonoid intermediate, EX, that lacks the proton of the bound amino acid. A pK value of 6.65 was determined indirectly for labilization of the proton of l-alanine, thus indicating the probable participation of an imidazole group of the enzyme in the labilization reaction. The tritium exchange reaction with tryptophan is shown to occur at rates faster than the over-all rate of pyruvate formation. ,ß elimination reactions catalyzed by tryptophanase thus occur in two distinct steps: elimination of an hydrogen from ES to form EX, followed by elimination of the ß substituent from EX to form an enzyme-aminoacrylate complex, EA. In conjunction with previous kinetic data, the results show that the ß elimination reaction per se is the rate-limiting step. These data thus support fully the mechanism shown in Equation 1 for these reactions. Partial structures for the intermediates are discussed. The pronounced spectral

EH (±H⁺)/ E (±S)/ ES (±H⁺)/ EX (±R^-)/ EA (-E//H₂O pyruvate + NH₃ (1)

changes that occur when Na⁺ replaces K⁺ in the ionic environment of holotryptophanase indicate important changes in conformation of the active site of the holoenzyme. Such changes are also indicated by differences in the rate of combination of apoenzyme with pyridoxal-P under the two sets of conditions.

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