2-Amino-3-ketobutyrate-CoA ligase from beef liver mitochondria. Purification and partial sequence.
2-Amino-3-ketobutyrate-CoA ligase (EC 2.3.1.29), or aminoacetone synthetase, has been purified by a nine-step procedure from 1.0 kg of beef liver to yield 8.8 mg of homogeneous enzyme. The homogeneous form of the enzyme, a monomer of M(r) = 44,000, shows unusually high absorption at 430 nm, with a ratio of absorbance at 280 and 430 nm of 2.6. On storage a species with an additional absorption peak at 332 is formed. Neither the 430-nm peak nor the 332-430 ratio is affected by pH or substrates. The peak at 430 nm and enzyme activity are both reduced by borohydride reduction and treatment with cysteine. The first 21 amino acids at the NH2-terminal of the ligase occur in the sequence Ser-Ala-Leu-Ala-Gln-Leu-Arg-Gly-Ile-Leu-Glu-Glu-Glu-Leu-Glu-Ser-Ile-Arg- Gly-Ala - Gly. No homology is detectable in the first 20 amino acids of the Escherichia coli and beef liver mitochondria enzymes. However, homology is found around the lysine residue to which the pyridoxal 5'-phosphate is attached in the two enzymes. A very hydrophobic peptide containing pyridoxal phosphate having the following sequence Leu-Leu-Gly-Val-Met-Asp-Gln-Val-Thr-Ile-Ile-Asn-Ser-Thr-Leu-Gly-Lys(P xy)-Ala- Leu-Gly-Gly-Ala-Ser-Gly-Gly-Tyr-Thr-Thr-Gly-Pro-Gly-Ala-Leu-Val has been isolated from the ligase. Fourteen residues around the lysine to which the pyridoxal 5'-phosphate is bound are completely identical with the pyridoxal 5'-phosphate containing peptide isolated from the E. coli 2-amino-3-ketobutyrate-CoA ligase.
Tong
H
H
Department of Chemistry, University of Iowa, Iowa City 52242.
Davis
L
L
D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D010455:Peptides; D000123:Acetyltransferases; C020679:glycine acetyltransferase