Intrinsic versus extrinsic stabilization of enzymes: the interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes.

摘要: We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due or factors leads decreased activity. A4-LDH a warm-temperate goby (Gillichthys mirabilis), which has lower values kcat Michaelis constant pyruvate ( K m PYR), was intrinsically more stable than Antarctic notothenioids Parachaenichthys charcoti Chionodraco rastrospinosus, as shown by higher apparent transition ('melting') (Tm(APP)). used four solutes, glycerol, sucrose, trimethylamine-N-oxide poly(ethylene glycol) 8000, stabilize proteins through modes preferential exclusion, study temperature-solute interactions three orthologs. Changes in Tm(APP) were similar all each solute tested, but catalytic rate G. mirabilis most increased temperature. In contrast, PYR affected that both conclude (a) exclusion functions within native state favor conformational microstates minimal surface area; (b) varied kinetic properties interaction between this nonspecific differing stabilities orthologs; (c) rates equally if measurements made at physiologically appropriate temperatures; (d) global stability localized flexibility these may evolve independently.