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.
Klink
T A
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Department of Biochemistry, University of Wisconsin-Madison, WI, USA.
Woycechowsky
K J
KJ
Taylor
K M
KM
Raines
R T
RT
D000818:Animals; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D003545:Cysteine