title: Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase: refined phosphorylation loop structure in the active site.
abstract: We report here that alterations of either His291-alpha or His146-beta' in the active site of human branched-chain alpha-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-alpha, 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 His291-alpha in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-beta', which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-alpha 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-beta' mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His291-alpha plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His146-beta' is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.
authors.affiliation: Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
authors.forename: R Max
authors.initials: RM
authors.lastname: Wynn
authors.forename: Mischa
authors.initials: M
authors.lastname: Machius
authors.forename: Jacinta L
authors.initials: JL
authors.lastname: Chuang
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Diana R
authors.initials: DR
authors.lastname: Tomchick
authors.forename: David T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D020134:Catalytic Domain; D019281:Dimerization; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D011522:Protons; D011768:Pyruvate Dehydrogenase Complex; D006639:Histidine; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D010100:Oxygen