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Figure 6
Stereoview of NAD binding by trypanosomal GAPDH (full black). For clarity
only 2 of the 4 subunits are shown. A substantial deviation of the protein backbone
occurs in human GAPDH (open gray, only one subunit shown) near the adenosine
part of the cofactor. The nearby cleft (marked by an asterisk) is important for
introducing selectivity in inhibitor binding and has therefore been termed “selectivity cleft.”
Page 374
glycosomal GAPDH has been overexpressed in E. coli [59] while human erythrocyte GAPDH is
available from commercial suppliers. The sequences of the two enzymes are only 55% identical [60].
The crystal structure of the parasite enzyme was solved from Laue data at 3.2 Å resolution in our group
(Figure 6) and in a second crystal form at 2.8 Å [26]. The structure of the human muscle enzyme was
solved at 3.5 Å resolution in the group of the late Herman Watson [28]. Its resolution was improved to
2.3 Å in our group [26,29]. Both structures were of the holo-enzyme, i.e., the enzyme in presence of the
cofactor.
The active site and the nicotinamide-binding site of the two enzymes are very well conserved. This is
reflected in similar K m (glyceraldehyde-3-phos-phate) values of 0.15 and 0.17 mM for the trypanosomal
and human enzyme, respectively [61]. Surprisingly, the K m (NAD +) values differ by a factor ten: 0.45
mM for T. brucei and 0.04 mM for human GAPDH [61]. In view of the conservation of the
nicotinamide and pyrophosphate binding sites, the substantial difference in K m (NAD +) has to be
ascribed to the adenosine binding environment. Indeed, some of the residues embracing the adenine ring
of the cofactor are not identical. In the trypanosomal enzyme the adenine is sandwiched in between a
Thr in the back and a Met in the front. This Met is replaced by Pro and Phe in the human enzyme
(Figure 7). A second difference involves the residue flanking the C2 atom of the purine ring. It is a Val
in the trypanosomal enzyme, and Asn in the human enzyme. These differences apparently account for a
ten-fold lower affinity for the cofactor.
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