Photoinactivation and photoaffinity labeling of tryptophan synthase .alpha.2.beta.2 complex by the product analog 6-azido-L-tryptophan

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Abstract
The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the .alpha.2.beta.2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (.lambda.max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 .mu.M). Photolysis of the quinonoid form of the .alpha.2.beta.2 complex results in time-dependent inactivation of the .beta.2 subunit but not of the .alpha. subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the .alpha.2.beta.2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified .alpha.2.beta.2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the .alpha. subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the .alpha.2.beta.2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.