Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate.
L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit. We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid. We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent. The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding. The stoichiometry of inactivation was 1 mol of BAPMP per subunit. We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with [14C]iodoacetic acid. The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.
Nakayama
T
T
Institute for Chemical Research, Kyoto University, Japan.
Esaki
N
N
Tanaka
H
H
Soda
K
K
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D003545:Cysteine; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D046911:Macromolecular Substances; D011733:Pyridoxamine; C017087:bromoacetylpyridoxamine phosphate; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D003545:Cysteine