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Characterization of the alpha-peptide released upon protease activation of pyruvate oxidase.
The pyruvate oxidase of Escherichia coli is a homo-tetrameric enzyme which can be activated greater than 500-fold (kcat/Km) by limited proteolytic digestion with alpha-chymotrypsin in the presence of pyruvate and thiamine pyrophosphate. The cleavage produces an Mr 2000 peptide (the alpha-peptide) from each subunit and mimics the physiologically important activation of the enzyme by phospholipids. Moreover, the proteolytic cleavage results in the loss of the high affinity lipid-binding site of the enzyme. We now report the isolation and characterization of the alpha-peptide fragment which is cleaved from the carboxyl terminus of each subunit by protease activation. Both the site of cleavage and the sequence of the alpha-peptide have been determined by a combination of Edman degradation of the purified peptide and DNA sequence analysis of the gene encoding the oxidase. The cleavage site lies within a sequence of hydrophobic amino acids predicted to form a beta-sheet. Another segment of the alpha-peptide is predicted to form an amphipathic alpha-helix. Quantitative assessment of the amphipathic nature of this alpha-helix (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623) gives a value very similar to the values for several helical peptides which spontaneously bind to the surface of phospholipid vesicles. From these analyses, we propose that the alpha-peptide may play a role in binding pyruvate oxidase to cell membrane phospholipids in vivo.
PMID: 3902830 [PubMed - indexed for MEDLINE]
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Cited by 3 PubMed Central articles
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Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli.
Neumann P, Weidner A, Pech A, Stubbs MT, Tittmann K.
Proc Natl Acad Sci U S A. 2008 Nov 11; 105(45):17390-5. Epub 2008 Nov 6.
[Proc Natl Acad Sci U S A. 2008]
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Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein.
Grabau C, Cronan JE Jr.
Nucleic Acids Res. 1986 Jul 11; 14(13):5449-60.
[Nucleic Acids Res. 1986]
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Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphipathic protein that controls phosphatidylcholine synthesis.
Kalmar GB, Kay RJ, Lachance A, Aebersold R, Cornell RB.
Proc Natl Acad Sci U S A. 1990 Aug; 87(16):6029-33.
[Proc Natl Acad Sci U S A. 1990]