Evidence for the presence of two kinetically distinct active forms of ribonuclease T2. The pH dependence of the steady-state kinetic parameter, kcat, for transphosphorylation of both a natural and a synthetic substrate.
A kinetic study has been made of the RNase-T2-catalyzed transphosphorylation of two adenine nucleotides, adenylyl(3'-5')uridine and adenosine 3'-(1-naphthyl)phosphate. Rates were measured at pH values ranging from 2.6 to 8.2. The observed shape of the plot of log kcat against pH for both the natural and the synthetic substrate suggests that there exist two parallel rate-determining pathways. Two pH-independent rate constants and three ionization constants of the enzyme-substrate complexes were obtained by nonlinear iterative least-squares analysis. Detailed interpretation of the pH profiles was carried out and it is proposed that carboxylate anion is likely to deprotonate O-2' at 4 less than pH less than 6, but at pH greater than 6 an alternative general base would play this role more effectively than the carboxylate group. Another base in its protonated cationic form is responsible for the general acid catalysis.
D001665:Binding Sites; D002021:Buffers; D004720:Endonucleases / Q000378:metabolism*; D004722:Endoribonucleases; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D012260:Ribonucleases / Q000378:metabolism*; D013379:Substrate Specificity
D002021:Buffers; D007527:Isoenzymes; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2)