A kinetic study of metabolite transfer in coupled two-enzyme reactions

Abstract: There are two fundamentally different ways by which a metabolite can be transferred between enzymes in metabolic pathways. If the two sequential enzymes cannot form a complex with each other, the metabolite must first be released from the producing enzyme to the reaction medium and then be transported to the consuming enzyme, via the reaction medium, by free diffusion. If the two sequential enzymes are capable of forming a complex with each other, an alternative metabolite transfer mechanism could be operative. The metabolite might then be directly transferred from the producing enzyme to the consuming one without prior release to solution. The term ?channelling? refer to such a mechanism of direct metabolite transfer. The mechanism by which this metabolite transfer takes place is a main determinant of the kinetic characteristics and control properties of the sequential metabolic enzyme reactions. Establishing what mechanism of metabolite transfer is applicable in specific cases, or in general, is therefore a matter of outstanding importance for our understanding of the behaviour and regulation of metabolic pathways. This thesis deals with the hypothesis concerning metabolite channelling in systems that may form dynamic bi-enzyme complexes in vitro. Some crucial steady-state and transient-state kinetic experiments were carried out, aiming at testing if there is any general difference in the mechanism of NADH transfer between dehydrogenases depending on the chiral coenzyme specificity of the enzymes. The next investigation was initiated to examine the unchallenged proposal of a channelled transfer of dihydroxyacetone phosphate in the reaction system of aldolase and glycerol 3-phosphate dehydrogenase, just as the unchallenged isotope dilution data taken to be indicative of a channelled transfer of glyceraldehyde-3-phosphate in the reaction system of aldolase and glyceraldehyde- 3-phosphate dehydrogenase. In the final two studies the coupled reactions catalysed by the fusion protein of malate dehydrogenase and citrate synthase, and of beta-galactosidase and galactose dehydrogenase were examined, by using a new and more direct approach where all predictions on the expected free diffusion behaviour of the fusion protein were based upon the kinetic properties of the fusion protein itself. From the results achieved it was concluded that no tenable evidence is available for metabolite channelling in systems involving glycolytic enzymes; and the results obtained also provide clear evidence that channelling due to proximity effects either is absent or of negligible quantitative significance.