Purification and characterization of the branched chain alpha-ketoacid dehydrogenase complex from Saccharomyces cerevisiae.
Branched chain alpha-ketoacid dehydrogenase complex was purified from Saccharomyces cerevisiae by polyethylene glycol fractionation and chromatography on Sephacryl S-200, DEAE-cellulose and Sepharose CL-2B. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated the enzyme contained subunits of M(r) = 57,000, 52,000, 47,000 and 38,000. The specific activity of the purified enzyme was 0.82 mumol NADH/min/mg protein at 30 degrees C with 16 mM alpha-ketoisovalerate as substrate. The apparent Km values for alpha-ketoisovalerate, alpha-ketoisocaproate and alpha-keto-beta-methylvalerate were 21, 22, and 20 mM, respectively. The preparation was also able to oxidize the intermediates of threonine and methionine metabolism, alpha-keto-gamma-methiolbutyrate and alpha-ketobutyrate, with Km values of 13 and 8 mM, respectively.
Sinclair
D A
DA
School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.
Dawes
I W
IW
Dickinson
J R
JR
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D045782:Hemiterpenes; D007651:Keto Acids; D009097:Multienzyme Complexes; C016211:alpha-keto-beta-methylvaleric acid; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)