AMINO-ACID SUBSTITUTIONS AT THE DIMER INTERFACE OF HUMAN GLUCOSE-6-PHOSPHATE-DEHYDROGENASE THAT INCREASE THERMOSTABILITY AND REDUCE THE STABILIZING EFFECT OF NADP

Over 100 mutations of the G6PD gene have been documented. With the con struction of the molecular model of glucose-6-phosphate dehydrogenase, based on the structure of the bacterial Leuconostoc mesenteroides glu cose-6-phosphate dehydrogenase, it has been possible to superimpose th ese amino acid changes on to the structure of the glucose-6-phosphate dehydrogenase molecule. There are a large number of severe disease cau sing mutations at the dimer interface which usually cause decreased th ermostability. We have used this knowledge to predict amino acid chang es which would effect an increase in the stability of the dimer. The a spartic acid at residue 421 was chosen as it is a negatively charged r esidue at the centre of the dimer interface in an area rich in negativ ely charged residues. This residue was changed to a neutrally charged alanine or asparagine, or a positively charged lysine or arginine. The thermostability of the enzyme was increased when residue 421 was neut ral (A or N) and increased further when positive (K or R). NADP is kno wn to exert a concentration dependent stabilising effect on the glucos e-6-phosphate dehydrogenase dimer. However the concentration-dependent stabilising effect of NADP was reduced in the residue-421 substitutio ns in a manner which was inversly proportional to charge change. These results suggest that changes at the dimer interface can also affect t he distant (> 20 Angstrom) NADP-binding site, and vice versa; an attem pt has been made to explain these interactions based on the molecular model of human glucose-6-phosphate dehydrogenase.