CATALYSIS OF THIOL/DISULFIDE EXCHANGE

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Disulfide bond formation is an integral part of the expression of numerous extracellular proteins including receptors, enzymes, and hormones. The mechanisms by which correct disulfide bonds are formed during protein folding and assembly is not only important to understanding how three-dimensional protein structure is generated from primary sequence information but also has practical significance in the expression and production of disulfide-containing proteins and peptides of therapeutic importance. Protein disulfide isomerase, an abundant protein of the endoplasmic reticulum, catalyzes thiol/disulfide oxidation, reduction, and rearrangement reactions involved in the oxidative folding of proteins. The long-range goal of the proposed research is to understand at a structural and mechanistic level how protein disulfide isomerase, either by itself or in conjunction with other proteins, facilitates the folding and assembly of proteins that contain disulfide crosslinks. Site-directed mutagenesis, kinetic methods, and protein chemistry will be applied to investigate the mechanism by which the three dithiol/disulfide centers of the enzyme participate individually or collectively in catalysis. The kinetic behavior of mutant enzymes in which one or more specific cysteine residues have been converted to Ser and Ala will be compared to the behavior of the wildtype protein to detect interactions between the multiple thiol/disulfide centers. Covalent and noncovalent interactions between protein disulfide isomerase and its protein substrates will be explored. Single cysteine mutants will be produced that may trap the enzyme in non-productive covalent complexes with the substrates. The participation of the enzyme in the formation of intermolecular and intramolecular disulfide bonds during the in vitro assembly of multichain proteins such as immunoglobulin G and its fragments (Fab) and the cooperation between protein disulfide isomerase and other assembly proteins such as the molecular chaperonins will -also be investigated.