Methionine formation from alpha-ketomethiobutyrate in the trypanosomatid Crithidia fasciculata.
Methionine consumed during the synthesis of polyamines can be recycled in most organisms by a unique pathway wherein the final step is the transaminative conversion of alpha-ketomethiobutyrate to methionine (KMAT activity). In the trypanosomatid Crithidia fasciculata, three separate aminotransferases (KMAT-A, -B, -T) were found to catalyse this activity. All three aminotransferases were found to utilise aromatic amino acids as the amino donor for the KMAT reaction, but KMAT-A functioned optimally with histidine and KMAT-B with arginine as amino donors. KMAT-T was found to operate best with aromatic amino acids and glutamate as amino donors, and was also found to catalyse aspartate aminotransferase and tyrosine aminotransferase activities. Amino acid sequencing of internal peptides from KMAT-T yielded a sequence with very high identity to vertebrate, cytosolic aspartate aminotransferase. As pig heart cytosolic aspartate and alanine aminotransferases were found to be unable to catalyse KMAT activity, the crithidial enzyme appears to be an aspartate aminotransferase with unusual catalytic properties. Inhibition studies on C. fasciculata homogenates showed that carboxymethoxylamine, canaline, and nitrophenylalanine were effective inhibitors of total KMAT activity (63-100% inhibition at 1 mM in the presence of 1 mM alpha-ketomethiobutyrate and 30 mM total amino acid as substrates) and the individual, isolated enzymes. At 1 mg ml-1, canaline was found to inhibit cell growth in vitro by 62%, and carboxymethoxylamine caused cell death within 24 h.
Berger
B J
BJ
Department of Biochemistry, University of Dundee, UK. bjberger@bad.dundee.ac.uk
Dai
W W
WW
Wilson
J
J
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D016831:Crithidia fasciculata / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
D002087:Butyrates; D004791:Enzyme Inhibitors; D001224:Aspartic Acid; D008715:Methionine; D000637:Transaminases; C114944:alpha-ketomethiobutyrate aminotransferase; D000409:Alanine