Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.
A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.
Farrenkopf
B C
BC
Carl A. Olson Chemistry Laboratories, Rutgers, State University, Newark, New Jersey 07102.
Jordan
F
F
D000494:Allosteric Regulation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D011767:Pyruvate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D012440:Saccharomyces / Q000201:enzymology*; D013329:Structure-Activity Relationship
D005656:Fungal Proteins; D007527:Isoenzymes; D011773:Pyruvates; D011767:Pyruvate Decarboxylase; C009144:pyruvamide