Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn2+

Jamie Jon Arnold, David W. Gohara, Craig Eugene Cameron

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3Dpol) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn 2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+.

Original languageEnglish (US)
Pages (from-to)5138-5148
Number of pages11
JournalBiochemistry
Volume43
Issue number18
DOIs
StatePublished - May 11 2004

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Ribonucleotides
RNA Replicase
Poliovirus
Nucleotides
Kinetics
Conformations
Enzymes
Divalent Cations
Substrates
Catalysis
Mental Competency
Metal ions
Cations
Metals
Binding Sites

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Arnold, Jamie Jon ; Gohara, David W. ; Cameron, Craig Eugene. / Poliovirus RNA-Dependent RNA Polymerase (3Dpol) : Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn2+. In: Biochemistry. 2004 ; Vol. 43, No. 18. pp. 5138-5148.
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abstract = "The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3Dpol) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn 2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+.",
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Poliovirus RNA-Dependent RNA Polymerase (3Dpol) : Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn2+. / Arnold, Jamie Jon; Gohara, David W.; Cameron, Craig Eugene.

In: Biochemistry, Vol. 43, No. 18, 11.05.2004, p. 5138-5148.

Research output: Contribution to journalArticle

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N2 - The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3Dpol) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn 2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+.

AB - The use of Mn2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3Dpol) in the presence of Mn2+. The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn 2+, the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn2+, it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg2+, highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg2+, this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg2+.

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