A highly active immobilized ribonuclease.
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Chemistry Techniques, Analytical↗
Biomedical subjects
K J Woycechowsky
Publications and source records attributed to K J Woycechowsky.
Native disulfide bond formation in proteins.
Native disulfide bond formation is critical for the proper folding of many proteins. Recent studies using newly identified protein oxidants, folding catalysts, and mutant cells provide insight into the mechanism of oxidative protein folding in vivo. This insight promises new strategies for more efficient protein production.
Animals↗
Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.
Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.
Animals↗
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.
Acid Phosphatase↗