Lactate dehydrogenases from an abyssal fish, a dogfish, a tidepool sculpin, and a mammal have been found to differ in their ability to bind substrate analog and coenzyme at varying temperatures and pressures. Affinities for a substrate analog are quite similar for each lactate dehydrogenase at their respective biological temperatures, suggesting temperature-dependent modification of enzyme-substrate binding for optimal function. Binding of coenzyme by the three ectothermic enzymes is less affected by changes in temperature than is coenzyme binding by the mammalian enzyme, and coenzyme binding by the abyssal fish enzyme is considerably less sensitive to high hydrostatic pressure than it is in the case of the other three lactate dehydrogenases. The total free energy change involved in binding coenzyme and substrate analog is only slightly higher for the endothermic than for the three ectothermic enzymes, but the enthalpic and entropic contributions are quite different. The ectotherms appear to have minimized the enthalpic contribution and hence minimized temperature effects on binding. The relationship between enthalpy and entropy for each of the binding interactions studied is a straight line of slope within the limits found by other workers for water-solute interactions and/or weak bond formation and is presumed to be a result of the conformational changes accompanying ligand binding. The contributions to binding of the AMP and nicotinamide subsites of the coenzyme binding site give a good estimate of many of the binding interactions of the coenzyme as a whole, and appear to compensate one another to maintain low AH and AS values for coenzyme binding to the ectothermic enzymes. This same type of compensation in volume change can be seen between the substrate and coenzyme binding sites for the abyssal fish lactate dehydrogenase, resulting in a net volume change very close to zero. The observed temperature and pressure effects on binding cannot be explained solely in terms of the types of weak bonds involved, and known homologies between dogfish and pig LDH make major differences between the active sites unlikely. Conformational changes occurring simultaneously with binding may be of considerable importance in modifying the observed responses to both temperature and pressure. / Science, Faculty of / Zoology, Department of / Graduate

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

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Lactate dehydrogenase was isolated in pure form from crude extract of the cestode Hymenoleois diminuta by heat treatment and column chromatography. The purified enzyme has a specific activity of 106 units per mg protein. The molecular weight of the purified protein was 75,000 as determined by Sephadex gel filtration and analytical ultracentrifugation. An equilibrium ultracentrifugation study suggests a subunit molecular weight of 39,000. From these data, a dimer form of the native enzyme is proposed.

Thesis--Veterinaer- og Lanbohøjskole, Copenhagen. / Summary in Danish. Bibliography: p. 199-[221].

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