Dwyer T M, Rao K S, Westover J B, Kim J J, Frerman F E
Department of Pediatrics, The University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 2001 Jan 5;276(1):133-8. doi: 10.1074/jbc.M007672200.
Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the gamma-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-proton of acetoacetyl-CoA by Arg-94 --> Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.
戊二酰辅酶A脱氢酶催化戊二酰辅酶A氧化脱羧生成巴豆酰辅酶A和二氧化碳。该蛋白质的遗传性缺陷会导致I型戊二酸血症，这是一种致命的神经系统疾病。戊二酰辅酶A脱氢酶是酰基辅酶A脱氢酶家族中唯一在底物酰基部分结合位点含有阳离子残基（精氨酸-94）的成员。晶体学研究表明，精氨酸-94与戊二酰辅酶A的γ-羧酸盐处于氢键距离内。将精氨酸-94替换为甘氨酸（一种致病突变）以及与精氨酸空间结构更接近的谷氨酰胺，会使突变脱氢酶的催化常数（kcat）降至野生型酶的2%至3%。这些突变脱氢酶对戊二酰辅酶A的米氏常数（Km）增加了10至16倍。不发生脱羧反应的替代底物己酰辅酶A和戊二酰胺辅酶A的稳态动力学常数受突变影响较小。后一种变化可能是由于空间和极性效应。不可氧化底物类似物3-硫代戊二酰辅酶A和乙酰乙酰辅酶A的解离常数不受突变影响。然而，从3-硫代戊二酰辅酶A夺取α-质子以形成与氧化黄素的电荷转移复合物受到严重限制。相比之下，精氨酸-94→谷氨酰胺突变脱氢酶夺取乙酰乙酰辅酶A的α-质子不受影响，生成的烯醇盐与氧化黄素形成电荷转移复合物。这些实验表明，精氨酸-94对戊二酰辅酶A的结合没有重大贡献。然而，精氨酸-94的电场可能稳定由戊二酰辅酶A和3-硫代戊二酰辅酶A的α-质子夺取产生的双阴离子，这两种物质都含有γ-羧酸盐。精氨酸-94也可能使戊二酰辅酶A和3-硫代戊二酰辅酶A定向以便夺取α-质子。
