title: Protonation of crotonyl-CoA dienolate by human glutaryl-CoA dehydrogenase occurs by solvent-derived protons.
abstract: 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.
authors.affiliation: Department of Pediatrics, University of Colorado Health Sciences Center, Aurora, Colorado 80010, USA.
authors.forename: K Sudhindra
authors.initials: KS
authors.lastname: Rao
authors.forename: Mark
authors.initials: M
authors.lastname: Albro
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Zirrolli
authors.forename: David
authors.initials: D
authors.lastname: Vander Velde
authors.forename: David N M
authors.initials: DN
authors.lastname: Jones
authors.forename: Frank E
authors.initials: FE
authors.lastname: Frerman
mesh_terms: 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*
chemical_list: D000214:Acyl Coenzyme A; D011522:Protons; D012997:Solvents; C010701:crotonyl-coenzyme A; D050770:Glutaryl-CoA Dehydrogenase