Kinetic Studies of Yeast PolyA Polymerase Indicate an Induced Fit Mechanism for Nucleotide Specificity
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- 作者：Paul B. Balbo ; Gretchen Meinke ; and Andrew Bohm
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 31, 2005
- 年：2005
- 卷：44
- 期：21
- 页码：7777 - 7786
- 全文大小：228K
- 年卷期：v.44,no.21(May 31, 2005)
- ISSN：1520-4995
文摘
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 studiesof the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction(An + ATP 
An+1 + PPi) is adequately described by a rapid equilibrium random mechanism. Severalthermodynamic parameters for the reaction were determined or calculated, including the overall equilibriumconstant (Keq = 84) and the apparent equilibrium constant of the internal step (Kint = 4) which involvesthe rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accountsfor nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of thesulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining forboth reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanismby which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism fornucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects forATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correctsubstrate.
An+1 + PPi) is adequately described by a rapid equilibrium random mechanism. Severalthermodynamic parameters for the reaction were determined or calculated, including the overall equilibriumconstant (Keq = 84) and the apparent equilibrium constant of the internal step (Kint = 4) which involvesthe rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accountsfor nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of thesulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining forboth reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanismby which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism fornucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects forATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correctsubstrate.