A small-molecule catalyst of protein folding in vitro and in vivo.
BACKGROUND
The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (Eo').


RESULTS
A synthetic small-molecule dithiol, (+/-)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an Eo' value of -0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo.


CONCLUSIONS
A small-molecule dithiol with a low thiol pKa value and high disulfide Eo' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo.
Woycechowsky
K J
KJ
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Wittrup
K D
KD
Raines
R T
RT
D000135:Acid Phosphatase / Q000378:metabolism; D002384:Catalysis; D003510:Cyclohexanes / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology; D013438:Sulfhydryl Compounds / Q000378:metabolism
C403499:1,2-bis(2-mercaptoacetamido)cyclohexane; D003510:Cyclohexanes; D004220:Disulfides; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic; D000135:Acid Phosphatase; D019704:Protein Disulfide-Isomerases