Roles of His291-α and His146-β′ in the Reductive Acylation Reaction Catalyzed by Human Branched-chain α-Ketoacid Dehydrogenase: Refined phosphorylation loop structure in the active site

Abstract

We report here that alterations of either His291-α or His146-β′ in the active site of human branched-chain α-ketoacid dehydrogenase (E1b) impede both the decarboxylation and the reductive acylation reactions catalyzed by E1b as well as the binding of cofactor thiamin diphosphate (ThDP). In a refined human E1b active-site structure, His291-α, which aligns with His407 in Escherichia coli pyruvate dehydrogenase and HiS263 in yeast transketolase, is on a largely ordered phosphorylation loop. The imidazole ring of His 291-α in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-β′, which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-α mutation results in a complete order-to-disorder transition of the loop region, which precludes the binding of the substrate lipoyl-bearing domain to E1b. The H146A-β′ mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His 291-α plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His 146-α is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.