Evidence for the Two Phosphate Binding Sites of an Analogue of the Thioacyl Intermediate for theTrypanosoma cruziGlyceraldehyde-3-phosphate Dehydrogenase-Catalyzed Reaction, from Its Crystal Structure

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate (GAP) into d-glycerate 1,3-bisphosphate (1,3-diPG) in the presence of NAD(+) and inorganic phosphate (P(i)). Within the active site, two anion-binding sites were ascribed to the binding of the C3 phosphate of GAP (P(s)) and to the binding of the attacking phosphate ion (P(i)). The role played by these two sites in the catalytic mechanism in connection with the functional role of coenzyme exchange (NADH-NAD(+) shuttle) has been investigated by several studies leading to the C3 phosphate flipping model proposed by Skarzynski et al. [Skarzynski, T., Moody, P. C., and Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187]. This model has not yet received direct confirmation. To gain further insight into the role of both sites, we synthesized irreversible inhibitors which form with the essential cysteine residue a thioacyl enzyme analogue of the catalytic intermediate. Here we report the refined glycosomal Trypanosoma cruzi GAPDH in complex with a covalently bound GAP analogue at an improved resolution of 2.0-2.5 A. For this holo-thioacyl enzyme complex, a flip-flop movement is clearly characterized, the change from the P(i) to the P(s) binding site being correlated with the coenzyme exchange step: the weaker interaction of the intermediate when bound at the P(s) site with the cofactor allows its release and also the binding of the inorganic phosphate for the next catalytic step. This result gives strong experimental support for the generally accepted flip-flop model of the catalytic mechanism in GAPDH.

Structure-Based Drug Discovery for Tropical Diseases

“…Therefore, the vital dependence on glycolysis as the main source of energy for the parasites, combined to distinct molecular properties when compared with their human counterparts makes the glycolytic enzymes attractive targets for drug design. Structure-and ligand-based approa- ches on enzymes of the carbohydrate metabolism pathway have identified several promising inhibitors, as well as shed light on the structural basis for selective inhibition [102][103][104][105][106][107][108][109][110][111][112][113][114][115][116][117].…”

Trapping of the Thioacylglyceraldehyde-3-phosphate Dehydrogenase Intermediate from Bacillus stearothermophilus

“…This result indicates that the redox reaction has occurred in the crystalline state, with the hydride transfer from C1 of G3P to C4 of nicotinamide ring and concom- The fractional reduction of NAD ϩ by G3P was calculated at different G3P concentrations by fitting the observed spectra to a linear combination of the spectrum of the native form and the arsenate-treated form. Inset: dependence of the fractional reduction of NAD ϩ as a function of G3P concentration (10,15,30, and 60 mM). Spectra recorded on different crystals were normalized using the spectrum of the holoenzyme as reference.…”

The Potential of Secondary Metabolites from Plants as Drugs or Leads against Protozoan Neglected Diseases—Part III: In-Silico Molecular Docking Investigations

“…1GYP [119]; 1A7K [120]; 1GYQ [121] 1YWG [122]; 2B4R, 2B4T [123] 2X0N [124]; 4P8R [125] 1K3T [126]; 1ML3 [127]; 1QXS [128]; 3IDS [129] Glycerol-3-phosphate dehydrogenase (GPDH) 1EVY, 1EVZ [130];1JDJ, 1M66, 1M67, 1N1G [131]; 1N1E [132] Glyoxalase I (GLO1) 2C21 [133] Glyoxalase II (GLO2) 2P18, 2P1E [134] GMP synthetase (GMPS) 3UOW [135] Guanylate kinase (GK) 1Z6G [136] Heat shock protein 90 (HSP90) 3H80 [137], 3Q5J, 3Q5K, 3Q5L, [138]; 3U67 [139] …”