2R0M

The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.

(2007) Biochemistry 46: 14468-14477

• PubMed: 18020372 Search on PubMed
• DOI: https://doi.org/10.1021/bi7009597
• Primary Citation of Related Structures:  
  2R0M, 2R0N


• PubMed Abstract: 
  

  We have determined steady-state rate constants and net rate constants for the chemical steps in the catalytic pathway catalyzed by the E370D mutant of glutaryl-CoA dehydrogenase and compared them with those of the wild-type dehydrogenase. We sought rationales for changes in these rate constants in the structure of the mutant cocrystallized with the alternate substrate, 4-nitrobutyric acid. Substitution of aspartate for E370, the catalytic base, results in a 24% decrease in the rate constant for proton abstraction at C-2 of 3-thiaglutaryl-CoA as the distance between C-2 of the ligand and the closest carboxyl oxygen at residue 370 increases from 2.9 A to 3.1 A. The net rate constant for flavin reduction due to hydride transfer from C-3 of the natural substrate, which includes proton abstraction at C-2, to N5 of the flavin decreases by 81% due to the mutation, although the distance increases only by 0.7 A. The intensities of charge-transfer bands associated with the enolate of 3-thiaglutaryl-CoA, the reductive half-reaction (reduced flavin with oxidized form of substrate), and the dienolate following decarboxylation are considerably diminished. Structural investigation suggests that the increased distance and the change in angle of the S-C1(=O)-C2 plane of the substrate with the isoalloxazine substantially alter rates of the reductive and oxidative half-reactions. This change in active site geometry also changes the position of protonation of the four carbon dienolate intermediate to produce kinetically favorable product, vinylacetyl-CoA, which is further isomerized to the thermodynamically stable normal product, crotonyl-CoA.

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Organizational Affiliation: 

Department of Pediatrics, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado 80045-0511, USA.