Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi.
A single form of serine hydroxymethyltransferase (SHMT) was detected in epimastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the enzyme characterized from another trypanosomatid, Crithidia fasciculata [Capelluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol. Biochem. Parasitol. 98, 187-201]. The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N-alpha-p-tosyl-L-lysine chloromethyl ketone and Ltrans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, however, the enzyme could be purified to homogeneity. Digitonin treatment of intact cells suggested that the enzyme is cytosolic. T. cruzi SHMT presents a monomeric structure shown by the apparent molecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephadex G-200 gel filtration and SDS/PAGE, respectively. This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate-dependent after L-cysteine and hydroxylamine treatments and it was strongly inhibited by the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards L-serine. Partial sequencing of tryptic internal peptides of the enzyme indicate considerable similarity with other SHMTs, particularly from those of plant origin.
Capelluto
D G
DG
Centro de Investigaciones Bioenergéticas, Facultad de Medicina-CONICET, Universidad de Buenos Aires, Argentina.
Hellman
U
U
Cazzulo
J J
JJ
Cannata
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D016831:Crithidia fasciculata / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D013045:Species Specificity; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D004791:Enzyme Inhibitors; D012696:Glycine Hydroxymethyltransferase