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

Paul B Balbo1, Gretchen Meinke, Andrew Bohm

  • 1Tufts University School of Medicine and Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.

Biochemistry
|May 25, 2005
PubMed

Insights

Polyadenylate polymerase (PAP) uses an induced fit mechanism to specifically add polyadenylate tails to mRNA. This mechanism ensures efficient ATP incorporation over other nucleotides.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Polyadenylate polymerase (PAP) is crucial for mRNA maturation, catalyzing the addition of polyadenylate tails.
  • Understanding PAP's reaction mechanism and substrate specificity is vital for molecular biology research.

Purpose of the Study:

  • To conduct a comprehensive kinetic analysis of Polyadenylate polymerase (PAP).
  • To elucidate the reaction mechanism and nucleotide specificity of PAP.

Main Methods:

  • Performed steady-state kinetic analysis, including initial velocity studies.
  • Conducted inhibition studies and experiments with alternative substrates.
  • Utilized sulfur elemental effect studies to probe rate-determining steps.

Main Results:

  • The PAP reaction follows a rapid equilibrium random mechanism.
  • A significant difference in Vmax (100-fold) dictates nucleotide specificity (ATP vs. CTP), not Km.
  • Kinetic data suggest an induced fit mechanism for substrate selection and catalysis.

Conclusions:

  • PAP employs a dual mechanism involving uniform binding and ground-state destabilization for ATP specificity.
  • The chemical step is rate-determining for both ATP and CTP reactions.
  • Kinetic parameters provide insights into the molecular basis of PAP's function in mRNA processing.

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