PRE-STEADY-STATE KINETIC-ANALYSIS OF 2-HYDROXY-6-KETO-NONA-2,4-DIENE-1,9-DIOIC ACID 5,6-HYDROLASE - KINETIC EVIDENCE FOR ENOL KETO TAUTOMERIZATION/

The reaction catalyzed by 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic ac id 5,6-hydrolase (MhpC) was analyzed by stopped-flow UV-visible kineti cs at 317 nm (substrate depletion) and 270 nm (product formation) at p H 5.0 and 4.0. Comparison of the rates and amplitudes of product forma tion versus substrate depletion provided evidence for the formation of a discrete keto-intermediate, as predicted from previous isotope exch ange experiments [Lam, W. W. Y., & Bugg, T. D. H. (1997) Biochemistry, 36, 12242-12251]. Accurate modeling of the concentration data could o nly be achieved using a branched kinetic mechanism in which the interm ediate is released at a rate comparable to its catalytic turnover, con sistent with the earlier isotope exchange data. The apparent ''leakine ss'' of the active site and relatively weak substrate binding (K-d = 3 0 mu M) are consistant with a mechanism in which the enzyme binds the dienol substrate in a strained, nonplanar conformation which promotes ketonization in the C-5 position to give a keto-intermediate.