Proton transfer in the mechanism of polyadenylate polymerase.
PAP (polyadenylate polymerase) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, the pH dependence of the steady-state kinetic parameters of yeast PAP were determined for the forward (adenyl transfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the forward reaction. The pH dependence of the maximum velocity of the forward reaction suggests two protonic species are involved in enzyme catalysis. Optimal enzyme activity requires one species to be protonated and the other deprotonated. The deuterium solvent isotope effect on Vmax is also consistent with proton transfer involved in catalysis of a rate-determining step. Finally, pKa calculations of PAP were performed by the MCCE (multiconformational continuum electrostatic) method. Together, the data support that the protonation of residues Lys215 and Tyr224 exhibit co-operativity that is important for MgATP2- and MgPPi2- binding/dissociation, and suggest these residues function in electrostatic, but not in general acid, catalysis.
Balbo
Paul B
PB
Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences, Department of Biochemistry, Boston, MA 02111, USA. pbalbo@gate.sinica.edu.tw
Bohm
Andrew
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D020134:Catalytic Domain; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011522:Protons; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology*
D005656:Fungal Proteins; D011522:Protons; D012313:RNA; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase