The project's goal is to use a new approach to simplify the kinetic and thermodynamic study of human erythrocyte glucose-6- phosphate dehydrogenase (G6PD) that involves the control of the enzyme's state of association. G6PD is a key enzyme in the hexosemonophosphate pathway and over 370 genetic variants of the human erythrocyte enzyme have been identified. Individuals with G6PD deficiencies can suffer hemolytic crisis on exposure to a variety of drugs or foods. The large number of genetic variants, many with reduced activity, mae G6PD an attractive target for cloning and sequencing. The interpretation of the changes in amino acid sequence are dependent on the measurement of G6PD's biochemical properties. Unfortunately, there is a lack of agreement on many of the basic enzymatic properties of erythrocyte G6PD, namely, the binding of substrates and the effect of intercellular molecules on the enzyme's kinetics. In analogy with prior work on yeast G6PD, this proposal's hypothesis is past kinetic and thermodynamic studies of human erythrocyte G6PD measured the properties of a mixture of G6PD dimers and tetramers, each of which may have different biochemical properties. This lack of agreement concerning human erythrocyte G6PD's biochemical properties would arise because the relative amounts of G6PD dimers and tetramers changed during the kinetic or thermodynamic measurements. Thus proposal will focus upon measuring the biochemical properties of the G6PD dimer, the form presumed to be present in the erythrocyte. The three specific aims of the RUI proposal are (1) to measure the number of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) molecules bound to G6PD and the magnitude of the association constant for these two molecules. The project will directly measure the equilibrium quantity of radioactively labeled G6P, or NADP, bound to the enzyme. The proposal will also (2) quantitatively study the inhibition of erythrocyte G6PD kinetics by ATP and 2,3 diphosphoglycerate, two proposed physiologically important inhibitors of G6PD. The third (3) specific aim will measure the dimertetramer association constant using large zone (plateau) HPLC experiments on a size exclusion column.
