Kinetic Mechanism of DNA Polymerase I (Klenow Fragment): Identification of a Second Conformational Change and Evaluation of the Internal Equilibrium Constant

Michael E. Dahlberg, Stephen Benkovic

Research output: Contribution to journalArticle

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Abstract

In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF′·DNAn+1·PPi·and KF·DNAn+1·PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P. Α., Johnson, Κ. Α., & Benkovic, S. J. (1987) Biochemistry 26, 8410–8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P. Α., & Benkovic, S. J. (1988) Biochemistry 27, 6716–6725]. In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF′·DNA·dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF′·DNAn+1·PPi and KF·DNAn+1·PPi.The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3′-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, Κ. Α., & Benkovic, S. J. (1985) Biochemistry 24, 4010–4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF·DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.

Original languageEnglish (US)
Pages (from-to)4835-4843
Number of pages9
JournalBiochemistry
Volume30
Issue number20
DOIs
StatePublished - May 1 1991

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DNA Polymerase I
Equilibrium constants
Biochemistry
Polymerization
Kinetics
Rate constants
Chemical bonds
DNA
Escherichia coli
Experiments
Enzymes
Direction compound

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{e35d4a4877424491ab3c5dbcba66f762,
title = "Kinetic Mechanism of DNA Polymerase I (Klenow Fragment): Identification of a Second Conformational Change and Evaluation of the Internal Equilibrium Constant",
abstract = "In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF′·DNAn+1·PPi·and KF·DNAn+1·PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P. Α., Johnson, Κ. Α., & Benkovic, S. J. (1987) Biochemistry 26, 8410–8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P. Α., & Benkovic, S. J. (1988) Biochemistry 27, 6716–6725]. In a pulse-chase experiment in this study, a burst amplitude of 100{\%} of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80{\%}, indicative of the accumulation of the KF′·DNA·dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF′·DNAn+1·PPi and KF·DNAn+1·PPi.The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3′-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, Κ. Α., & Benkovic, S. J. (1985) Biochemistry 24, 4010–4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF·DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.",
author = "Dahlberg, {Michael E.} and Stephen Benkovic",
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Kinetic Mechanism of DNA Polymerase I (Klenow Fragment) : Identification of a Second Conformational Change and Evaluation of the Internal Equilibrium Constant. / Dahlberg, Michael E.; Benkovic, Stephen.

In: Biochemistry, Vol. 30, No. 20, 01.05.1991, p. 4835-4843.

Research output: Contribution to journalArticle

TY - JOUR

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T2 - Identification of a Second Conformational Change and Evaluation of the Internal Equilibrium Constant

AU - Dahlberg, Michael E.

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AB - In a previously determined minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment (KF) of Escherichia coli DNA polymerase I, a nonchemical step that interconverted the KF′·DNAn+1·PPi·and KF·DNAn+1·PPi complexes was not observed in correct incorporation [Kuchta, R. D., Mizrahi, V., Benkovic, P. Α., Johnson, Κ. Α., & Benkovic, S. J. (1987) Biochemistry 26, 8410–8417] but was detected in misincorporation [Kuchta, R. D., Benkovic, P. Α., & Benkovic, S. J. (1988) Biochemistry 27, 6716–6725]. In a pulse-chase experiment in this study, a burst amplitude of 100% of the enzyme concentration is observed; under pulse-quench conditions, the burst amplitude is 80%, indicative of the accumulation of the KF′·DNA·dNTP species owing to a slow step subsequent to chemical bond formation. This latter step was unequivocally identified by single-turnover pyrophosphorolysis and pyrophosphate-exchange experiments as one interconverting KF′·DNAn+1·PPi and KF·DNAn+1·PPi.The rate constants for this step in both directions were established through the rate constants for processive synthesis and pyrophosphorolysis. Pyrophosphorolysis of a 3′-phosphorothioate DNA duplex confirmed that the large elemental effect observed previously [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, Κ. Α., & Benkovic, S. J. (1985) Biochemistry 24, 4010–4018] in this direction but not in polymerization is due to a marked decrease in the affinity of KF for the phosphorothioate-substituted duplex and not to the chemical step. The combination of the experimentally measured equilibrium constant for the bound KF·DNA species with the collective kinetic measurements further extends previous insights into the dynamics of the polymerization process catalyzed by KF.

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