Influence of the structure of water on the hydrolysis of cytidine 2',3'-phosphate catalysed by bovine pancreatic ribonuclease A.
The study of the temperature dependence of the hydrolysis of cytidine 2',3'-phosphate by bovine pancreatic ribonuclease A (EC 3.1.27.5) at pH 7.0 by using the pH-stat method showed a transition at 4 degrees C [J. A. Biosca and C. M. Cuchillo (1980) Biochem. J. 189, 655-657]. The breaks found in the Van't Hoff and Arrhenius plots at pH 7.0 were confirmed in the present work by following the reaction spectrophotometrically with a stopped-flow spectrophotometer adapted to the use of sub-zero temperatures. Similar results were found at pH 5.5. In addition it was found that the discontinuity disappears when 40% ethyleneglycol is present in the reaction mixture. However, in this latter instance a discontinuity around 0 degrees C appears in the Arrhenius plot. To explore the possibility that all these effects were due to a conformational transition in the protein, thermal perturbation experiments were carried out with the enzyme. A change in the slope of the plot of delta A290 as a function of temperature was found around 6 degrees C at pH 7.0 but not at pH 5.5. The results reported here can be interpreted as due to a change in the protein structure induced by the change of the structure of water. The studies carried out in the presence of ethyleneglycol also open the way to the cryoenzymological experimentation on ribonuclease.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D003563:Cyclic CMP / Q000378:metabolism*; D003597:Cytosine Nucleotides / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D005026:Ethylene Glycols; D006868:Hydrolysis; D010179:Pancreas / Q000201:enzymology; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000378:metabolism*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D014867:Water
D003597:Cytosine Nucleotides; D005026:Ethylene Glycols; D014867:Water; D003563:Cyclic CMP; C019310:cifostodine; D004720:Endonucleases; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic