SMALL MOLECULE CATALYST OF PROTEIN FOLDING

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Protein disulfide isomerase (PDI) catalyzed the interchange of disulfide bonds in substrate proteins. The two Cys-Gly-His-Cys active sites provide a thiol of low pKa and a disulfide bond of high reduction potential (E??). Trans-1,20bis(2-mercaptoacetamido)cyclohexane (BMC) mimics the pKa and E?? of the PDI active sites. In vitro assays demonstrate that BMC is an efficient catalyst of the reactivation of scrambled ribonuclease (Rnase) A, a substrate with non-native disulfide bonds. In vivo assays show that BMC in the growth medium of yeast cells increases the secretion of native acid phosphatase, which has eight disulfide bonds. The effect is similar to that from the overproduction of PDI in the yeast cells. A monothiol analog of BMC, N-methyl-mercaptoacetamide (NMA), also catalyzes the reactivation of scrambled Rnase A, but less efficiently than does BMC. These data indicate that molecules with a low thiol pKa and high disulfide E?? catalyze effectively the formation of native disulfide bonds in proteins. Moreover, BMC is a useful catalyst for the in vitro and in vivo folding of proteins that contain multiple disulfide bonds.