J. Biol. Chem., Vol. 269, Issue 6, 4057-4064, Feb, 1994
2-Amino-3-ketobutyrate-CoA ligase from beef liver mitochondria. Purification and partial sequence
H Tong and L Davis
Department of Chemistry, University of Iowa, Iowa City 52242.
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.
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