It is proposed to examine the active site structure of the protein disulfide oxidoreductase thioredoxin in an attempt to dissect the mechanism of this important and widespread enzyme. Preliminary structural characterization of the reduced form of thioredoxin has been completed using data from NMR spectroscopic methods, followed by distance geometry and restrained molecular dynamics calculations. The pH dependence of the NMR spectrum has also been studied and has given valuable new insights into the mechanism of disulfide reduction by thioredoxin. The studies reveal that one of the thiol groups in the active site of reduced (dithiol) thioredoxin titrates with a low pKa. In addition, an aspartate residue (Asp 26) close to the active site titrates with abnormally high pKa in the oxidized (disulfide) form of the protein, and probably in the reduced form as well. Due to the close spatial proximity of the three possible titrating groups (the thiols of Cys 32 and Cys 35 and the Asp 26 carboxyl group) in reduced thioredoxin, it is impossible to separate on e titration from another with any certainty: studies with mutant proteins are proposed in order to identify the titrating groups. The proton transfer effects in the active site region have considerable relevance to the mechanism of thioredoxin, for example, at neutral pH, the low-pKa thiol group (thought to be that of Cys 32) would be present in significant proportions as the thiolate, which may be a contributing factor in the observed fast rate of protein disulfide reduction by thioredoxin. The present proposal concentrates on a dissection of the active site, using NMR spectroscopic and structural methods. Solution structures of high accuracy are now possible using new NMR techniques incorporating stable isotope labels, which can be used to generate greatly expanded sets of distance and dihedral angle constraints for use in structure calculations. The generation of solution structures of the highest precision possible for both oxidized and reduced thioredoxins is an important part of this proposal. Carefully-chosen mutants will be employed to discover the significant features in the active site which give rise to the characteristic pH dependence and activity of thioredoxin. In addition, a comparison of the results of studies with mutants in which the active site sequences correspond to those of related proteins glutaredoxin and protein disulfide isomerase (PDI) should give valuable information on the sources of differences in reactivity and specificity between the enzymes in this important family. The potential exists in the thioredoxin system for a detailed study at the molecular level of the elements of structure in the active site region which influence the mechanism of this important enzymic reaction.

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National Institute of General Medical Sciences (NIGMS)
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