Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu.
Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue. The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected. The modified peptide was unstable under mildly acid or mildly basic conditions. Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue. Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography. Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively. Given the instability of the modification, it may be more prevalent than recognized hitherto. Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.
Rose
K
K
Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Simona
M G
MG
Savoy
L A
LA
Regamey
P O
PO
Green
B N
BN
Clore
G M
GM
Gronenborn
A M
AM
Wingfield
P T
PT
D000595:Amino Acid Sequence; D010583:Bacteriophage mu / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010659:Phenylhydrazines / Q000737:chemistry; D011773:Pyruvates / Q000378:metabolism*; D019289:Pyruvic Acid; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010659:Phenylhydrazines; D011773:Pyruvates; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C004787:2,4-dinitrophenylhydrazine; D019289:Pyruvic Acid