The oxidative folding rate of bovine pancreatic ribonuclease is enhanced by a covalently attached oligosaccharide.
Bovine pancreatic ribonuclease B (RNase B) differs from RNase A by the presence of an oligosaccharide moiety covalently attached to Asn 34. Oxidative folding studies of RNase B were carried out at different temperatures using DTT(ox) as the oxidizing agent, and the results were compared with those for RNase A. The oxidative folding rates of RNase B are between 1.7 and 1.3 times faster than those of RNase A at the temperatures that were investigated. The folding pathways of RNase B were determined to be similar to those of RNase A in that two structured intermediates, each lacking one native disulfide bond, were found to populate the regeneration pathways at 25 degrees C and pH 8.3. The thermodynamic stabilities of these two glycosylated intermediates, and their rates of formation from their unstructured precursors in the rate-determining step, were found to be higher than those of their unglycosylated counterparts from RNase A. Thus, the underlying cause for the faster rate of oxidative regeneration of native RNase B appears to be both thermodynamic and kinetic due to the higher stability, and faster rate of formation, of the intermediates of RNase B compared to those of RNase A.
Xu
Guoqiang
G
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Narayan
Mahesh
M
Scheraga
Harold A
HA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry*; D010084:Oxidation-Reduction / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004220:Disulfides; D007527:Isoenzymes; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D012259:Ribonuclease, Pancreatic; C504335:pancreatic ribonuclease B, bovine; D003545:Cysteine