Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
This enzyme catalyses the reversible oxidation and phosphorylation of glyceraldehyde-phosphate to 1,3-bisphosphoglycerate. All Trypanosomatidae studied, including T. brucei, have two separate isoenzymes, one in the cytosol and one in the glycosome. The glycosomal enzyme represents 0.5 % of all the cellular protein in the bloodstream form and 14 % of the glycosomal protein. The enzyme is homotetrameric with a molecular mass of 139 000 and a pI of 9.3 (Misset et al., 1986). Two tandemly linked identical genes encode a polypeptide of 358 amino acids with a calculated mass of 38.9 kDa (Michels et al., 1986) and 52-57 % identity with other GAPDH proteins. The polypeptide has several specific insertions that are conserved in all glycosomal GAPDHs and a C-terminal extension carrying a PTS1 import signal (Hannaert et al., submitted). The enzyme is inhibited by the epoxide-containing GAPDH inhibitor pentalenolactone and by suramin, gossypol and agaricic acid (Lambeir et al., 1991). Although its substrate-binding site is well conserved, it has been possible to synthesise epoxide or alpha-enone containing analogues that selectively and irreversibly inhibit not only the enzyme but also the growth of the trypanosome in an in vitro culture system (Willson et al., 1994). The binding site for the NAD cofactor has some interesting differences that lead to a reduced affinity for this cofactor. The enzyme has been crystallised and its three-dimensional structure solved (Vellieux et al., 1993). By the method of modelling and rational drug design a number of highly selective adenosine analogues have been synthesized that tightly fit the NAD binding pocket of the trypanosome enzyme, but do not bind to the corresponding pocket of the mammalian homologue (Verlinde et al., 1994; Van Calenbergh et al., 1995). Some of these compounds have a high selectivity for the glycosomal enzyme and inhibit its activity at sub-micromolar concentrations (Gelb et al., unpublished).
The cytosolic isoenzyme differs in subunit mass (36 kDa) and pI (6.2) from the glycosomal enzyme (Misset et al., 1987) and lacks the typical insertions and extension of the glycosomal enzyme. Comparison of the sequences of the two enzymes has revealed that they are only distantly related (Michels et al., 1991) and that one of them, most likely the cytosolic enzyme which is absent from bodonine and the euglenozoan organisms, must have entered the ancestral trypanosomatid by an event of horizontal gene transfer (Wiemer et al., 1995a). The function of this cytosolic isoenzyme is not clear. It could be involved, together with the cytosolic PGK, in the equilibration of the cytosolic redox potential with the phosphate potential, or be involved in the conversion of GA3P produced in the pentose-phosphate pathway to glycerate 3-phosphate.