Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons.
The protonation of crotonyl-CoA dienolate following decarboxylation of glutaconyl-CoA by glutaryl-CoA dehydrogenase was investigated. Although it is generally held that the active sites of acyl-CoA dehydrogenases are desolvated when substrate binds, recent evidence has established that water has access to the active site in these binary complexes of glutaryl-CoA dehydrogenase. The present investigation shows that the dehydrogenase catalyzes (a) a rapid exchange of C-4 methyl protons of crotonyl-CoA with bulk solvent and (b) protonation of crotonyl-CoA dienolate by solvent-derived protons under single turnover conditions. Both of the reactions require the catalytic base, Glu370. These findings indicate that decarboxylation proceeds via a dienolate intermediate. The involvement of water in catalysis by glutaryl-CoA dehydrogenase was previously unrecognized and is in conflict with a classically held intramolecular 1,3-prototropic shift for protonation of crotonyl-CoA dienolate.
Rao
K Sudhindra
KS
Department of Pediatrics, University of Colorado Health Sciences Center, Aurora, Colorado 80010, USA.
Albro
Mark
M
Zirrolli
Joseph A
JA
Vander Velde
David
D
Jones
David N M
DN
Frerman
Frank E
FE
D000214:Acyl Coenzyme A / Q000737:chemistry*; D050770:Glutaryl-CoA Dehydrogenase / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D012997:Solvents / Q000737:chemistry*
D000214:Acyl Coenzyme A; D011522:Protons; D012997:Solvents; C010701:crotonyl-coenzyme A; D050770:Glutaryl-CoA Dehydrogenase