Minimum requirements for protease activation of flavin pyruvate oxidase.
Previous investigations have shown that the catalytic efficiency (kcat/KM) of pyruvate oxidase can be enhanced 450-fold by chymotryptic cleavage of a 23-residue peptide (alpha-peptide) from the carboxy terminus of the enzyme. The minimum requirement for proteolytic activation has been investigated by exposing pyruvate oxidase to a variety of carboxypeptidases, either singly or in combination. The extent of carboxypeptidase hydrolysis was followed by analyzing the release of amino acids and by mass spectral analysis of the truncated alpha-peptides which were derived from the carboxypeptidase-treated preparations. The results indicate that the removal of 7 carboxy-terminal residues does not activate the enzyme whereas the removal of 10 or 11 residues produces activated pyruvate oxidase. Activation of pyruvate oxidase by endoproteinase Glu-C confirms the carboxypeptidase results. Endoproteinase Glu-C specificity predicts hydrolytic cleavage of the peptide bond between Glu-561 and Val-562 with the removal of 11 residues from the carboxy terminus of the enzyme.
Bertagnolli
B L
BL
Roger Adams Laboratory, Department of Biochemistry, University of Illinois, Champaign-Urbana 61801.
Hager
L P
LP
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D011771:Pyruvate Oxidase / Q000187:drug effects / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013057:Spectrum Analysis
D010455:Peptides; D005182:Flavin-Adenine Dinucleotide; D011771:Pyruvate Oxidase; D002268:Carboxypeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase