Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.
Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.
D000096:Acetone / Q000031:analogs & derivatives / Q000378:metabolism; D000123:Acetyltransferases / Q000032:analysis / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism
D000596:Amino Acids; D008670:Metals; D013438:Sulfhydryl Compounds; D000096:Acetone; D013912:Threonine; D011732:Pyridoxal Phosphate; D000123:Acetyltransferases; C020679:glycine acetyltransferase; C006495:aminoacetone