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 acid 5,6-hydrolase (MhpC) was analyzed by stopped-flow UV-visible kinetics at 317 nm (substrate depletion) and 270 nm (product formation) at pH 5.0 and 4.0. Comparison of the rates and amplitudes of product formation versus substrate depletion provided evidence for the formation of a discrete keto-intermediate, as predicted from previous isotope exchange experiments [Lam, W. W. Y., & Bugg, T. D. H. (1997) Biochemistry, 36, 12242-12251]. Accurate modeling of the concentration data could only be achieved using a branched kinetic mechanism in which the intermediate is released at a rate comparable to its catalytic turnover, consistent with the earlier isotope exchange data. The apparent "leakiness" of the active site and relatively weak substrate binding (Kd = 30 microM) are consistent 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.
Henderson
I M
IM
Department of Chemistry, University of Southampton, Highfield, U.K.
Bugg
T D
TD
D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity
D006899:Mixed Function Oxygenases; C019483:methylhydroxypyridine-carboxylate dioxygenase