Conformation coupled enzyme catalysis

Single-molecule and transient kinetics investigation of dihydrofolate reductase

Nina Goodey, R. Derike Smiley, Stephen J. Benkovic, Gordon G. Hammes

Research output: Contribution to journalArticleResearchpeer-review

91 Citations (Scopus)

Abstract

Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50% fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 Å. These results are correlated with other measurements related to conformation coupled catalysis.

Original languageEnglish
Pages (from-to)16835-16843
Number of pages9
JournalBiochemistry
Volume44
Issue number51
DOIs
StatePublished - 27 Dec 2005

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Tetrahydrofolate Dehydrogenase
Catalysis
Conformations
Molecules
Kinetics
Enzymes
Fluorescence Resonance Energy Transfer
Fluorescence
Hydrides
Rate constants
Catalyst activity
Amino Acids
Mutagenesis
Fluorescence microscopy
Substrates
Site-Directed Mutagenesis
NADP
Fluorescent Dyes
Fluorescence Microscopy
Isotopes

Cite this

Goodey, Nina ; Smiley, R. Derike ; Benkovic, Stephen J. ; Hammes, Gordon G. / Conformation coupled enzyme catalysis : Single-molecule and transient kinetics investigation of dihydrofolate reductase. In: Biochemistry. 2005 ; Vol. 44, No. 51. pp. 16835-16843.
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abstract = "Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50{\%} fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 {\AA}. These results are correlated with other measurements related to conformation coupled catalysis.",
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Conformation coupled enzyme catalysis : Single-molecule and transient kinetics investigation of dihydrofolate reductase. / Goodey, Nina; Smiley, R. Derike; Benkovic, Stephen J.; Hammes, Gordon G.

In: Biochemistry, Vol. 44, No. 51, 27.12.2005, p. 16835-16843.

Research output: Contribution to journalArticleResearchpeer-review

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T2 - Single-molecule and transient kinetics investigation of dihydrofolate reductase

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