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II. Three Glycolytic Enzymes Of T. Brucei: Molecular Biology, Biochemistry, and X-Ray
Crystallography
A. Triosephosphate Isomerase (TIM)
Page 371
TIM is a homodimeric enzyme that interconverts dihydroxyacetone phosphate and glyceraldehyde-3phosphate.
It ensures that both trioses derived from glucose can be used for ATP production in the
glycolytic pathway. Triosephosphate isomerase does not require any cofactor. Both the T. brucei and
human enzymes have been overexpressed in Escherichia coli [50,25] and their crystal structures were
solved in our group [24,25]. In addition, the structures of T. brucei TIM in complex with seven
nonselective competitive inhibitors, with inhibition constants of 300 μM or higher were determined:
monohydrogen phosphate [51], 2-phosphoglycerate [52], 3-phosphoglycerate [53], 3phosphonopropionate
[53], glycerol-3-phosphate [53], 2-(N-formyl-N-hydroxyamino)-ethyl phosphonic
acid [54], and N-hydroxy-4-phosphonobutanamide [55]. These studies gave an excellent picture of
different ligand binding modes and of the conformational flexibility of the enzyme.
All ligands interact with the main features of the catalytic machinery of the enzyme (Figure 3): (1) the
phosphate is sequestered by the positive end of a 3 10-helix and Lys13; (2) polar groups on the carbon
framework interact with His95 and Glu167, the catalytic electrophile and the catalytic base of the
enzyme, respectively; (3) the entire inhibitor is shielded from the bulk solvent by a flexible loop, which
normally closes over the substrate during catalysis to prevent phosphate elimination [56] (Figure 4). The
only exception to flexible loop closure is N-hydroxy-4-phosphono-butanamide. It binds to the enzyme
with the flexible loop in the open conformation because its size precludes loop closure. Thus, the
crystallographic binding studies point out that it should be possible to design two very different classes
of selective inhibitors: a class that binds to the enzyme in the closed loop conformation and one that
binds to the open loop conformation.
Selective inhibitor design in the case of TIM appears to be a formidable task. All residues within 10 Å
of the active site are conserved [25]. This is also reflected in the similarity of the kinetic characteristics
between trypanosomal and human TIM: for T. brucei TIM, K m (glyceraldehyde-3-phosphate) = 0.25
mM, kcat = 3.7 × 10 5min -1 [57]; for human TIM, K m = 0.49 mM, kcat = 2.7 × 10 5 min -1 [25]. There are
significant differences in the surface protein of the two enzymes about 15 Å away from the substrate
phosphorus atom [58]. In a shallow cleft, T. bruceiTIM has Ala100-Tyr101, while the human
counterpart of these residues is His-Val (Figure 5). The cleft is formed by the flexible loop of one
subunit of the enzyme and a different loop originating from another subunit. When the flexible loop
changes its conformation from the closed to the open form the cleft widens substantially. Moreover, the
Ala-Tyr dipeptide becomes then directly accessible from the active site, the distance being about
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