Intrinsic versus extrinsic stabilization of enzymes - The interaction of solutes and temperature on A(4)-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes

We examined the effects of temperature and stabilizing solutes on A(4)-lact ate dehydrogenase (A(4)-LDH) from warm- and cold-adapted fishes, to determi ne how extrinsic stabilizers affect orthologs with different intrinsic stab ilities. Conformational changes during substrate binding are rate-limiting for A(4)-LDH, thus stabilization due to intrinsic or extrinsic factors lead s to decreased activity. A(4)-LDH from a warm-temperate goby (Gillichthys m irabilis), which has lower values for k(cat) and the Michaelis constant for pyruvate (K-m(PYR)), was intrinsically more stable than the orthologs of t he cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chion odraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (T-m(APP)). We used four solutes, glycerol, sucrose, trimethyl amine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins thro ugh different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in T-m(APP) were similar for a ll orthologs in each solute tested, but the catalytic rate of G. mirabilis A(4)-LDH was decreased most by solutes and increased most by temperature. I n contrast, the K-m(PYR) values of the Antarctic orthologs were more affect ed than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A(4)-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the diffe ring intrinsic stabilities of the orthologs; (c) the catalytic rates of A(4 )-LDH orthologs are equally affected by stabilizing solutes, if measurement s are made at physiologically appropriate temperatures; and (d) global stab ility and localized flexibility of these A(4)-LDH orthologs may evolve inde pendently.