Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi.
Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.
Lowe
S E
SE
Department of Biochemistry, Michigan State University, East Lansing 48824.
Zeikus
J G
JG
D000595:Amino Acid Sequence; D002712:Chlorides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006038:Glyoxylates / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011767:Pyruvate Decarboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012505:Sarcina / Q000201:enzymology* / Q000254:growth & development; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D002712:Chlorides; D006038:Glyoxylates; D017967:Zinc Compounds; C016837:zinc chloride; D011767:Pyruvate Decarboxylase; D015032:Zinc; C031150:glyoxylic acid