Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

Abstract

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

“…Samples were evaluated for TcP5CDH and markers by both Western blotting and biochemical activity assays. Western blot analysis showed that TcTAT, a cytosolic marker (48), was released at digitonin concentrations ranging from 0.1 to 5 mg ml Ϫ1 (Fig. 9B), whereas TcGAPDH, a glycosomal marker, was released at intermediate digitonin concentrations (0.5-3 mg ml…”

Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases

“…2). The T. cruzi enzyme recognizes all three aromatic amino acids quite well and utilizes oxaloacetate, 2-ketoglutarate, and pyruvate (the favored substrate) as alternative amino acceptors (40). This broad-specificity enzyme would seem to be a strong candidate for a contemporary ''dinosaur'' similar to the common ancestor of the superfamily.…”

Crystallization and preliminary X-ray analysis of tyrosine aminotransferase from Trypanosoma cruzi epimastigotes

“…TAT from T. cruzi (Tulahuen strain, stock Tul 2) epimastigotes was purified to homogeneity by chromatography on DEAE celullose, gel filtration on Sephacryl S-200 and chromatography on MonoQ with a fast protein liquid chromatography system as described previously (Montemartini et al, 1993). For crystallization experiments the enzyme was brought to a protein concentration of 3-4 mg ml -~ using Centricon concentrators.…”

“…2.6.1.5; TAT) from T~panosoma cruzi, the causative agent of American trypanosomiasis (Chagas' disease) has been deduced from the cDNA sequence (Bontempi et al, 1993;Montemartini, Santome, Cazzulo & Nowicki, 1993). The enzyme is 416 residues long and shows only 30% sequence homology to ASAT and TAT from E. coli but approximately 77% homology (Bontempi et al, 1993) to the glucocorticoid inducible rat (Grange et al, 1985) and human liver (Rettenmeier, Natt, Zentgraf & Scherer, 1990) TAT's.…”

“…Unlike mammalian TAT's, which transaminate only aromatic amino acids with o~-ketoglutarate, the parasite enzyme is able to accept the three ot-ketoacids; pyruvic, o~-ketoglutaric and oxaloacetic, the three aromatic amino acids and alanine as substrate. The best substrate pairs appear to be tyrosine-pyruvic acid and alanine--~-ketoglutaric acid (Montemartini et al, 1993).…”