ANION BINDING AT THE ACTIVE-SITE OF TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE - MONOHYDROGEN PHOSPHATE DOES NOT MIMIC SULFATE

The three-dimensional structure of triosephosphate isomerase complexed with the competitive inhibitor HPO4(2-) was determined by X-ray crystallography to a resolution of 0.24 nm. A comparison with the native crystal structure, where SO4(2-) is bound, revealed five changes: (a) a 0.10-nm shift of the anion-binding site; (b) a further closing of the flexible loop of the enzyme; (c) a 'swinging in' of the side chain of the catalytic Glu, that is chi-1 changes from (+) to (-) synclinal; (d) an altered water structure; (e) a disappearance of the conformational heterogeneity at the C-terminus of strand beta-7. Some of these changes may be related to the different hydrogen-bond pattern about the two different anions. However, the distance of 0.10 nm between the sulphur and phosphorus positions is unexpected and remains intriguing

CRYSTALLOGRAPHIC AND MOLECULAR MODELING STUDIES ON TRYPANOSOMAL TRIOSEPHOSPHATE ISOMERASE - A CRITICAL-ASSESSMENT OF THE PREDICTED AND OBSERVED STRUCTURES OF THE COMPLEX WITH 2-PHOSPHOGLYCERATE

In the continuation of a project aimed at the rational design of drugs against diseases caused by trypanosomes, the crystal structure of trypanosomal triosephosphate isomerase in complex with the active site inhibitor 2-phosphoglycerate has been determined. Two alternative modeling protocols have been attempted to predict the mode of binding of this ligand. In the first protocol, certain key interactions were restrained in the modeling procedure. In the second protocol, a full search of ligand conformational space was performed. In both cases the protein scaffold was kept static. Both protocols produced models which were reasonably close to the observed structure (rms difference <2.0 angstrom). Nevertheless, some essential features were missed by each of the protocols. The crystallographic structure of the 2-PGA TIM complex shows that the ligand binds fully within the active site of TIM, with partners for all but one of the ligand's strongly hydrogen bonding groups. Several of the interactions between the ligand and the active site of TIM are seen to be common to all of the complexes so far structurally characterized between trypanosomal triosephosphate isomerase and competitive inhibitors. Such key interactions appear to be the best guide in the prediction of the binding mode of a new inhibitor

Synthesis and Structure-activity-relationships of Analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a Selective Inhibitor of Trypanosomal Glycosomal Glyceraldehyde-3-phosphate Dehydrogenase

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD: GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized. Some of them displayed an interesting improvement in inhibitory activity compared to 1. Carbocyclic or acyclic analogs showed marked loss of activity, illustrating the importance of the typical (C-2'-endo) puckering of the ribose moiety. We also describe the synthesis of a pair of compounds that combine the beneficial effects of a 2- and 8-substituted adenine moiety on potency with the beneficial effect of a 2'-amido moiety on selectivity. Unfortunately, in both cases, IC50 values demonstrate the incompatibility of these combined modifications. Finally, introduction bf a hydrophobic 5'-amido group on 5'-deoxyadenosine enhances the inhibition of the protozoan enzyme significantly, although the gain in selectivity is mediocre

Vampire grades: can simulations support learning without reflection?

Experiential learning through computer simulation is claimed to involve reflection. This study examines the relationship between reflection and assessment using self report, simulation performance over time and summative assessment for 107 students. No relationship between reflection and summative assessment was found. Expected stimuli for reflection were negatively related with assessment