3-hydroxy-3-methylglutaryl-coenzyme A synthase reaction intermediates: detection of a covalent tetrahedral adduct by differential isotope shift 13C nuclear magnetic resonance spectroscopy.
Binding of [1,2-(13)C]acetyl-CoA to wild-type 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is characterized by large upfield shifts for C1 (184 ppm, Deltadelta = 20 ppm) and C2 (26 ppm, Deltadelta = 7 ppm) resonances that are attributable to formation of the covalent [1,2 -(13)C]acetyl-S-enzyme reaction intermediate. NMR spectra of [1, 2-(13)C]acetyl-S-enzyme prepared in H(2)(16)O versus H(2)(18)O indicate a 0.055 ppm upfield shift of the C1 resonance in the presence of the heavier isotope. The magnitude of this (18)O-induced (13)C shift suggests that the 184 ppm resonance is attributable to a reaction intermediate in which C1 exhibits substantial carbonyl character. No significant shift of the C2 resonance occurs. These observations suggest that, in the absence of second substrate (acetoacetyl-CoA), enzymatic addition of H(2)(18)O to the C1 carbonyl of acetyl-S-enzyme occurs to transiently produce a tetrahedral species. This tetrahedral adduct exchanges oxygen upon backward collapse to re-form the sp(2)-hybridized thioester carbonyl. In contrast with HMG-CoA synthase, C378G Zoogloea ramigera beta-ketothiolase, which also forms a (13)C NMR-observable covalent acetyl-enzyme species, exhibits no (18)O-induced shift. Formation of the [(13)C]acetyl-S-enzyme reaction intermediate of HMG-CoA synthase in D(2)O versus H(2)O is characterized by a time-dependent isotope-induced upfield shift of the C1 resonance (maximal shift = 0. 185 ppm) in the presence of the heavier isotope. A more modest upfield shift (0.080 ppm) is observed for C378G Z. ramigera beta-ketothiolase in similar experiments. The slow kinetics for the development of the deuterium-induced (13)C shift in the HMG-CoA synthase experiments suggest a specific interaction (hydrogen bond) with a slowly exchangeable proton (deuteron) of a side chain/backbone of an amino acid residue at the active site.
Vinarov
D A
DA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Miziorko
H M
HM
D000105:Acetyl Coenzyme A / Q000138:chemical synthesis; D000102:Acetyl-CoA C-Acyltransferase / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000138:chemical synthesis; D000818:Animals; D001665:Binding Sites; D001717:Birds; D002247:Carbon Isotopes; D003903:Deuterium / Q000737:chemistry; D004795:Enzyme Stability; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000138:chemical synthesis / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010103:Oxygen Isotopes; D012997:Solvents; D014867:Water / Q000737:chemistry
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D012997:Solvents; D014867:Water; C010667:acetoacetyl CoA; D000105:Acetyl Coenzyme A; D003903:Deuterium; D000102:Acetyl-CoA C-Acyltransferase; D006904:Hydroxymethylglutaryl-CoA Synthase