Kinetic mechanism of glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase (GCD) is a homotetrameric enzyme containing one noncovalently bound FAD per monomer that oxidatively decarboxylates glutaryl-CoA to crotonyl-CoA and CO2. GCD belongs to the family of acyl-CoA dehydrogenases that are evolutionarily conserved in their sequence, structure, and function. However, there are differences in the kinetic mechanisms among the different acyl-CoA dehydrogenases. One of the unanswered aspects is that of the rate-determining step in the steady-state turnover of GCD. In the present investigation, the major rate-determining step is identified to be the release of crotonyl-CoA product because the chemical steps and reoxidation of reduced FAD are much faster than the turnover of the wild-type GCD. Other steps are only partially rate-determining. This conclusion is based on the transit times of the individual reactions occurring in the active site of GCD.

Alternate substrates of human glutaryl-CoA dehydrogenase: structure and reactivity of substrates, and identification of a novel 2-enoyl-CoA product.

The dehydrogenation reaction catalyzed by human glutaryl-CoA dehydrogenase was investigated using a series of alternate substrates. These substrates have various substituents at the gamma position in place of the carboxylate of the physiological substrate, glutaryl-CoA. The steady-state kinetic constants of the six alternate substrates and the extent of flavin reduction in the anaerobic half-reaction were determined. One of these substrates, 4-nitrobutyryl-CoA, was previously thought not to be a substrate of the dehydrogenase; however, the enzyme does oxidize this substrate analogue with a k(cat) that is less than 2% of that with glutaryl-CoA when ferrocenium hexafluorophosphate (FcPF(6)) is the electron acceptor. Anaerobic titration of the dehydrogenase with 4-nitrobutyryl-CoA showed no reduction of the flavin; but instead showed an increased absorbance in the 460 nm region suggesting deprotonation of the analogue to form the alpha-carbanion. Analysis of these data indicated a binding stoichiometry of about 1.0. Under aerobic conditions, a second absorption maximum is observed with lambda(max) = 366 nm. The generation of the latter chromophore is dependent on an electron acceptor, either O(2) or FcPF(6), and is greatly facilitated by the catalytic base Glu370. The 466 nm absorbing species remains enzyme-bound while the 366 nm absorbing species is present only in solution. The latter compound was identified as 4-nitronate-but-2-enoyl-CoA by mass spectrometry, (1)H NMR, and chemical analyses. Ionization of the enzymatic product, 4-nitro-but-2-enoyl-CoA, that yields the nitronate occurs in solution and not on the enzyme. The variation of k(cat) with the nature of the substituent suggests that the various substituents affect the free energy of activation, Delta G(++), for dehydrogenation. There is a good correlation between log(k(cat)) and F, the field effect parameter, of the gamma-substituent. No correlation was found between any other kinetic or equilibrium constants and the substituent parameters using quantitative structure-activity relationships (QSAR). 4-Nitrobutyryl-CoA is the extreme example with the strongly electron-withdrawing nitro group in the gamma position.