Isolation and partial characterization of a broad specificity aminotransferase from Leishmania mexicana promastigotes.
A broad specificity aminotransferase (BSAT), with high activity with both, aromatic amino acids and aspartate as substrates, was purified to homogeneity from promastigotes of Leishmania mexicana by a method involving chromatography on DEAE-cellulose, Red-120-Sepharose and Mono Q, and gel filtration on Sephacryl S-200. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, was 90 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequence of four internal peptides obtained by tryptic digestion. Two of these peptides, consisting of 49 amino acid residues in total, showed high similarity (57%) with corresponding sequences of plant aspartate aminotransferases, whereas they had only 33% identity with the aromatic aminotransferase of Escherichia coli, and 16% identity with the tyrosine aminotransferase from the related parasite Trypanosoma cruzi. The BSAT contained only one 1/2 Cys residue per monomer. The optimal pH for the enzyme reaction, with tyrosine and alpha-oxoglutarate as substrates, was 7.0. The apparent Km values for tyrosine, phenylalanine, tryptophan and glutamate, with oxaloacetate as co-substrate, were 1.3, 0.9, 0.9 and 171.8 mM, respectively; the value for aspartate with alpha-oxoglutarate as co-substrate was 2.5 mM, and that for alanine with alpha-oxoglutarate as co-substrate was 216 mM. The values for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as co-substrate, were 5.6, 0.71 and 0.12 mM, respectively. These results suggest that the enzyme is a broad-specificity aminotransferase, able to transaminate the aromatic amino acids, aspartate, and to a lower extent alanine, with high sequence similarity to aspartate aminotransferases.
Vernal
J
J
IQUIFIB (CONICET-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Cazzulo
J J
JJ
Nowicki
C
C
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013379:Substrate Specificity; D000637:Transaminases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000596:Amino Acids; D000637:Transaminases