KINETICS AND THERMODYNAMIC STUDIES OF NADP BINDING REACTIONS OF GENETIC VARIANTS OF HUMAN ERYTHROCYTE GLUCOSE 6-PHOSPHATE DEHYDROGENASE

Abstract

Inherited Glucose-6-Phosphate dehydrogenase (G6PD) deficiency in humans results in hemolytic anaemia. The enzyme G6PD provides a crucial link in a series of biochemical reactions which occur in the red blood cell that leads to the steady state accumulation of NADPH, reduced glutathione by glutathione reductase, and the removal of potentially dangerous organic peroxides which, if not scanvenged, may result in the formation of radical species of oxygen which damage the energy generation system, which in turn may result in swelling, lysis and hemolytic anaemia. The objective of this thesis was to investigate the human variants of the enzyme G6PD in order to provide a better understanding of the molecular basis of the enzyme activity and factors affecting the onset of the human disease. Human Erythrocyte Glucose-6- Phosphate dehydrogenase has been known to occur in many genetic variants and the catalytic active enzyme of each variant are tetramers and dimers in acidic and alkaline solution respectively. The question then is whether there would be differences in the reactivities of these variants and whether there are differences in the reactivities of the two active forms of the enzyme from the same variant. A comparative analysis of the kinetic and thermodynamic studies of NADP+ binding reactions of these variants under controlled and well-defined experimental conditions of pH and ionic strength was therefore undertaken. The binding reaction of NADP+ to G6PD B+ was also studied as a function of ionic strength of the buffers in order to evaluate the effect of these variations in the buffer system on the co-operative interactions of the NADP+ binding sites on the enzyme. The findings show that there are two binding sites on each of the enzyme variants and these were identified as imidazolium groups of histidine and sulfhydryl groups. The logKm versus pH curves show a broad plateau between pH 6.7 and 8.2 interrupted by a sharp minimum at pH 7.1 for all the enzyme variants. An explanation of this behaviour in terms of co-operative ionization of groups on the enzyme and enzyme-substrate complex which may be linked to the association - dissociation behaviour of the enzyme is proposed. In agreement with G6P binding data, the plot of the enthalpy of the dissociation of enzyme - NADP+ complex against pH shows the shape of a two U—shaped curves consistent with the existence of a tetrameric form of enzyme at acidic pH and dimeric form at alkaline pH. A similar plot of the activation energy of the reaction for each variant shows a consistent decrease of the activation energy with increase in pH, the activation energy at the pH 5.8 being almost halved at the alkaline pH of 9.0, This behaviour is explained to arise from the dimer enzyme being more reactive than the tetramer. There are two schools of thought about the existence and nature of cooperativity among the NADP+ binding sites on G6PD subunits. The study reported in this thesis has unequivocally solved the controversy between the two schools. It is now established that the tetrameric form of the enzyme shows no Cooperativity while the dimeric form is cooperative. The disagreement between the two schools of thought has been explained in the variable experimental conditions used by the workers in the two schools. We have shown that an experimental condition that favours tetramer formation therefore favours non-cooperativity while a condition that favours dimer formation favours cooperativity. The inhibition study by primaquine phosphate shows a complex interaction of this effector with G6PD. There is activation of the G6PD activity at low effector concentration and inhibition at high concentration. This interaction may be due to oxidation of NADPH at low primaquine concentration resulting in generation of more NADP+ which increases the activity of the enzyme. Such a situation might account for the increased hemolysis in variant subjects with low Artracellular NADPH concentration which will result in low level of reduced glutathione. Reduced glutathione is necessary for the maintenance of the integrity of the red cells.