Kinetic characterization of inosine monophosphate dehydrogenase of Leishmania donovani.
Trypanosomatid protozoan pathogens are purine auxotrophs that are highly dependent on the enzyme inosine monophosphate dehydrogenase (IMPDH) for the synthesis of guanylate nucleotides. Enzymatic characterization of the Leishmania donovani IMPDH (LdIMPDH) overexpressed in E. coli revealed that this enzyme was highly specific for the substrates IMP and NAD(+) with K(m)(app) values of 33 and 390 microM, respectively. In contrast to other IMPDHs, LdIMPDH exhibits no substrate inhibition in high concentrations of NAD(+). Kinetic studies revealed that XMP and GMP were inhibitors with K(i) values of approximately 26 and 210 microM, respectively, suggesting that these nucleotides may regulate LdIMPDH activity. Mycophenolic acid was also a potent inhibitor of L. donovani IMPDH with a K(i) value of approximately 25 nM. Confocal immunofluorescence microscopy and subcellular fractionation localized LdIMPDH to the glycosome. Protein-protein interaction assays revealed that LdIMPDH associated tightly with glycosomal protein sorting receptor LdPEX5.
Dobie
Fredrick
F
Institute of Parasitology, Macdonald Campus of McGill University, 21, 111 Lakeshore Road, Ste. Anne-de-Bellevue, Quebec, Canada H9X 3V9.
Berg
Amanda
A
Boitz
Jan M
JM
Jardim
Armando
A
D000595:Amino Acid Sequence; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006157:Guanosine Monophosphate / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007893:Leishmania donovani / Q000737:chemistry / Q000201:enzymology*; D008830:Microbodies / Q000737:chemistry; D018613:Microscopy, Confocal; D009173:Mycophenolic Acid / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D000074435:Peroxisome-Targeting Signal 1 Receptor; D010802:Phylogeny; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D016415:Sequence Alignment; D013347:Subcellular Fractions; D013379:Substrate Specificity; D019820:Xanthine
D004791:Enzyme Inhibitors; D000074435:Peroxisome-Targeting Signal 1 Receptor; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D012265:Ribonucleotides; D009243:NAD; D007291:Inosine Monophosphate; D019820:Xanthine; C011141:xanthosine monophosphate; D006157:Guanosine Monophosphate; D009173:Mycophenolic Acid