Role of the variable active site residues in the function of thioredoxin family oxidoreductases.

The enzymes of the thioredoxin family fulfill a wide range of physiological functions. Although they possess a similar CXYC active site motif, with identical environment and stereochemical properties, the redox potential and pK(a) of the cysteine pair varies widely across the family. As a consequence, each family member promotes oxidation or reduction reactions, or even isomerization reactions. The analysis of the three-dimensional structures gives no clues to identify the molecular source for the different active site properties. Therefore, we carried out a set of quantum mechanical calculations in active site models to gain more understanding on the elusive molecular-level origin of the differentiation of the properties across the family. The obtained results, together with earlier quantum mechanical calculations performed in our laboratories, gave rise to a consistent line of evidence, which points to the fact that both active site cysteines play an important role in the differentiation. In contrary to what was assumed, differentiation is not achieved through a different stabilization of the solvent exposed cysteine but, instead, through a fine tuning of the nucleophilicity of both active site cysteines. Reductant enzymes have both cysteine thiolates poorly stabilized, oxidant proteins have both cysteine thiolates highly stabilized, and isomerases have one thiolate (solvent exposed) poorly stabilized and the other (buried) thiolate highly stabilized. The feasibility of shifting the chemical equilibrium toward oxidation, reduction, or isomerization only through subtle electrostatic effects is quite unusual, and it relies on the inherent thermoneutrality of the catalytic steps carried out by a set of chemically equivalent entities all of which are cysteine thiolates. Such pattern of stabilization/destabilization, detected in our calculations is fully consistent with the observed physiological roles of this family of enzymes. 2008 Wiley Periodicals, Inc.