ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 352, No. 1, April 1, pp. 114–120, 1998 Article No. BB980577 3-Hydroxy-3-methyl-glutaryl-CoA Reductase in Trypanosoma ( Schizotrypanum) cruzi: Subcellular Localization and Kinetic Properties Juan L. Concepcion,* Dolores Gonzalez-Pacanowska,† and Julio A. Urbina‡ ,1 *Laboratorio de Enzimologı B a de Parasitos, Facultad de Ciencias, Universidad de Los Andes, Me ´rida, Venezuela; †Instituto de Parasitologı B a y Biomedicina ‘‘Lo ´pez Neyra,’’ Consejo Superior de Investigaciones Cientificas, c/Ventanilla 11, 18001 Granada, Spain; and ‡Laboratorio de Quimica Biolo ´gica, Centro de Bioquı B mica y Biofı B sica, Instituto Venezolano de Investigaciones Cientı B ficas, Apartado 21827, Caracas 1020A, Venezuela Received October 17, 1997, and in revised form December 23, 1997 gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH 2 -termi- The subcellular localization of 3-hydroxy-3-methyl- nal membrane-spanning sequence. This is the first glutaryl-CoA (HMG-CoA) reductase, which catalyzes demonstration of a soluble eukaryotic HMG-CoA re- the first committed step of the mevalonate pathway, ductase and also the first report on the presence of was investigated in Trypanosoma cruzi epimastigotes an enzyme of the isoprenoid biosynthesis pathway in using well-established cell fractionation procedures. glycosomes.  1998 Academic Press It was found that ca. 80% of the activity of the enzyme Key Words: Trypanosoma cruzi; epimastigotes; sterol was associated with the glycosomes, microbody-like biosynthesis; 3-hydroxy-3-methyl-glutaryl-CoA reduc- organelles unique to kinetoplastid protozoa which tase; glycosomes; digitonin-permeabilized cells. contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity asso- ciated with microsomes. The glycosome-associated en- zyme is not membrane-bound as it was recovered The protozoan parasites Trypanosoma (Schizotrypa- quantitatively in the aqueous phase of the biphasic num) cruzi and the different species of the Leishmania system formed by Triton X-114 at 30°C. Studies with genus require, as fungi and yeasts, the presence of spe- digitonin-permeabilized intact epimastigotes demon- cific endogenous sterols for cell viability and prolifera- strated the presence of two pools of soluble HMG-CoA tion (1–10); this fact has led to interest in the develop- reductase in these cells, associated to the cytoplasmic ment of sterol biosynthesis inhibitors (SBI 2 ), which are and glycosomal compartments. Steady-state kinetic currently the mainstay of antifungal chemotherapy studies of the glycosome-associated enzyme indicated (11 – 13), as antiparasitic agents (14, 15). The sterol classical Michaelis – Menten behavior with K m,app biosynthesis pathways of several of these organisms (HMG-CoA) 28 { 3 mM, K m,app (NADPH) 37 { 4 mM, and have been inferred from the analysis of sterol composi- V m,app 3.9 { 0.2 nmol/min mg protein; the transition- tion in the absence and presence of SBI’s, which act at state analog lovastatin behaved as a competitive inhib- different points of the pathway (2–5, 8, 9). However, itor with respect to HMG-CoA with K is 23 nM and a in contrast to fungi and yeasts (16, 17), practically noncompetitive inhibitor toward NADPH with K ii 29 nothing is known about the kinetic, molecular, and ge- nM. The results are in complete agreement with recent 2 Abbreviations used: SBI, sterol biosynthesis inhibitors; HMG- 1 To whom correspondence should be addressed at Current ad- dress: Department of Chemistry, University of Illinois at Urbana – CoA, 3-hydroxy-3-methyl-CoA reductase; PMSF, phenylmethylsulfo- nyl fluoride; TLCK, N-a-p-tosyl-L-lysine chloromethyl ketone; ASAT, Champaign, Chemistry and Life Sciences Laboratory, Room A106, 600 S. Mathews Avenue, Urbana, IL 61801. Fax: (217) 244-0997. E- alanine amino-transferase; PEPCK, phosphoenolpyruvate carboxy- kinase; PGI, phospho-glucose isomerase. mail: jaurbina@churchill.scs.uiuc.edu. 114 0003-9861/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.