Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A.
Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.
D000818:Animals; D002417:Cattle; D004951:Esterification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011072:Poly U / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013379:Substrate Specificity; D014500:Uracil Nucleotides / Q000378:metabolism*
D009841:Oligonucleotides; D009843:Oligoribonucleotides; D014500:Uracil Nucleotides; C027916:oligo(U); D011072:Poly U; D012259:Ribonuclease, Pancreatic