Intrinsic versus extrinsic stabilization of enzymes

• Published in: European Journal of Biochemistry
• Main Authors: Fields, Peter A., Wahlstrand, Benjamin D., Somero, George N.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2001
• Subjects: Antarctic; The Antarctic; Antarc*
• Online Access: http://dx.doi.org/10.1046/j.1432-1327.2001.02374.x; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1046%2Fj.1432-1327.2001.02374.x; https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.2001.02374.x

We examined the effects of temperature and stabilizing solutes on A 4 ‐lactate dehydrogenase (A 4 ‐LDH) from warm‐ and cold‐adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate‐limiting for A 4 ‐LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A 4 ‐LDH from a warm‐temperate goby ( Gillichthys mirabilis ), which has lower values for k cat and the Michaelis constant for pyruvate ( ), was intrinsically more stable than the orthologs of the cold‐adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus , as shown by a higher apparent transition (‘melting’) temperature ( T m(APP) ). We used four solutes, glycerol, sucrose, trimethylamine‐ N ‐oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature–solute interactions of the three orthologs. Changes in T m(APP) were similar for all 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. In contrast, the values of the Antarctic orthologs were more affected 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 differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A 4 ‐LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A 4 ‐LDH orthologs may evolve independently.