2R0M

The effect of a Glu370Asp Mutation in Glutaryl-CoA Dehydrogenase on Proton Transfer to the Dienolate Intermediate

Summary for 2R0M

• Entry DOI: 10.2210/pdb2r0m/pdb
• Related: 2r0n
• Descriptor: Glutaryl-CoA dehydrogenase, FLAVIN-ADENINE DINUCLEOTIDE, 4-nitrobutanoic acid, ... (4 entities in total)
• Functional Keywords: glutaryl-coa dehydrogenase, flavoprotein, isomerase, alternative splicing, disease mutation, fad, mitochondrion, oxidoreductase, polymorphism, transit peptide
• Biological source: Homo sapiens (human)
• Cellular location: Mitochondrion matrix: Q92947
• Total number of polymer chains: 1
• Total formula weight: 44421.33

Authors

Rao, K.S.,Fu, Z.,Albro, M.,Narayanan, B.,Baddam, S.,Lee, H.J.,Kim, J.J.,Frerman, F.E. (deposition date: 2007-08-20, release date: 2008-04-22, Last modification date: 2023-08-30)

Primary citation

Rao, K.S.,Fu, Z.,Albro, M.,Narayanan, B.,Baddam, S.,Lee, H.J.,Kim, J.J.,Frerman, F.E.
The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.
Biochemistry, 46:14468-14477, 2007

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.

PubMed: 18020372
DOI: 10.1021/bi7009597

Experimental method

X-RAY DIFFRACTION (2.7 Å)