The subunit structure of alpha-acetohydroxyacid isomeroreductase from Salmonella typhimurium.
Alpha-Acetohydroxyacid isomeroreductase from Salmonella typhimurium has a native molecular weight of 220,000. The constituent polypeptide chains exhibit anomalous but unimodal electrophoretic migration on sodium dodecyl sulfate-urea polyacrylamide gels. The subunit molecular weight, determined by sedimentation equilibrium in 6 M guanidine hydrochloride, is 57,000. The apparent tetrameric nature of the native enzyme was confirmed by determining the types of oligomers formed upon cross-linking with dimethylsebacimidate. Analysis of tryptic peptides suggests that the polypeptide chains have an identical amino acid sequence. Carbohydrate analysis, ultraviolet absorption spectrum, and atomic absorption spectrum are consistent with the lack of cobalamine and cobalt. The Michaelis constants are as follows: alpha-acetolactate, 2.9 x 10-4 M; alpha-aceto-alpha-hydroxybutyrate, 7.8 x 10-4 M; NADPH, 1.5 x 10-5 M; Mg2+, 7.7 x 10-4 M. The catalytic constants (molecules of substrate catalyzed per min per molecules of enzyme) for alpha-acetolactate and alpha-aceto-alpha-hydroxybutyrate are 1,100 and 4,700, respectively. Comparative tryptic peptide analysis and immunological analysis show that alpha-acetohydroxyacid isomero-reductase and biosynthetic L-threonine deaminase bear no structural relationship and therefore rule out a "shared structure" hypothesis for the putative involvement of L-threonine deaminase in the synthesis of alpha-acetohydroxyacid isomeroreductase.
D000429:Alcohol Oxidoreductases / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006146:Guanidines; D006880:Hydroxy Acids; D005779:Immunodiffusion; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits / Q000276:immunology; D012486:Salmonella typhimurium / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014461:Ultracentrifugation; D014631:Valerates
D000596:Amino Acids; D006146:Guanidines; D006880:Hydroxy Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014631:Valerates; D000429:Alcohol Oxidoreductases; D014357:Trypsin; D008274:Magnesium