Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity.

Polyadenylate polymerase (PAP) catalyzes the synthesis of 3'-polyadenylate tails onto mRNA. A comprehensive steady-state kinetic analysis of PAP was conducted which included initial velocity studies of the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction (A(n) + ATP <--> A(n+1) + PP(i)) is adequately described by a rapid equilibrium random mechanism. Several thermodynamic parameters for the reaction were determined or calculated, including the overall equilibrium constant (K(eq) = 84) and the apparent equilibrium constant of the internal step (K(int) = 4) which involves the rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accounts for nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of the sulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining for both reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanism by which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism for nucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects for ATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correct substrate.