Rao K Sudhindra, Albro Mark, Zirrolli Joseph A, Vander Velde David, Jones David N M, Frerman Frank E
Department of Pediatrics, University of Colorado Health Sciences Center, Aurora, Colorado 80010, USA.
Biochemistry. 2005 Oct 25;44(42):13932-40. doi: 10.1021/bi050525+.
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.
研究了戊二酰辅酶A脱氢酶使戊二酰辅酶A脱羧后巴豆酰辅酶A二烯醇化物的质子化过程。尽管通常认为当底物结合时酰基辅酶A脱氢酶的活性位点是去溶剂化的，但最近的证据表明，在戊二酰辅酶A脱氢酶的这些二元复合物中，水可以进入活性位点。本研究表明，该脱氢酶催化：（a）巴豆酰辅酶A的C-4甲基质子与大量溶剂的快速交换；（b）在单周转条件下，溶剂衍生的质子使巴豆酰辅酶A二烯醇化物质子化。这两个反应都需要催化碱Glu370。这些发现表明脱羧反应通过二烯醇化物中间体进行。戊二酰辅酶A脱氢酶催化过程中水的参与此前未被认识到，并且与经典的分子内1,3-质子转移使巴豆酰辅酶A二烯醇化物质子化的观点相矛盾。
