Paul B Balbo, Gretchen Meinke, Andrew Bohm
Tufts University School of Medicine and Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.
Biochemistry 2005 May 31Polyadenylate 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.
Paul B Balbo, Gretchen Meinke, Andrew Bohm. Kinetic studies of yeast polyA polymerase indicate an induced fit mechanism for nucleotide specificity. Biochemistry. 2005 May 31;44(21):7777-86
PMID: 15909992
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