Purification and partial structural and kinetic characterization of an aromatic L-alpha-hydroxy acid dehydrogenase from epimastigotes of Trypanosoma cruzi.
An aromatic L-alpha-hydroxyacid dehydrogenase (AHADH) was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl-Sepharose and affinity chromatography on Affi-Gel Blue. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is about 80 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequences of 4 internal peptides obtained by CNBr cleavage at Met residues, and one peptide obtained after tryptic digestion. Three of the peptides presented considerable sequence similarity with the corresponding sequences of several malate dehydrogenases. The optimal pH for the enzyme reaction with p-hydroxyphenyl pyruvate and NADH as substrates was 7.5; that for the reverse reaction was 9.5. The apparent Km values for phenylpyruvate and p-hydroxyphenyl-pyruvate were 48 and 117 microM, respectively; that for L-phenyllactate in the reverse reaction was 420 microM. The enzyme was much less active with alpha-isocaproic acid as substrate, and other acids, including pyruvic and oxaloacetic, were not substrates at all. L-phenyllactic acid, but not the D-isomer, acted as substrate. The enzyme can therefore be considered as a general aromatic L-alpha-hydroxyacid dehydrogenase. The low apparent Km value for NADH (25 microM in the presence of phenylpyruvate) makes AHADH a candidate for the reoxidation of cytosolic NADH in T. cruzi.
Montemartini
M
M
IQUIFIB (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Santomé
J A
JA
Cazzulo
J J
JJ
Nowicki
C
C
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D045022:Lactate Dehydrogenases; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000596:Amino Acids; D000429:Alcohol Oxidoreductases; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; C021379:D-2-hydroxyacid dehydrogenase