Effect of osmolytes on protein dynamics in the lactate dehydrogenase- catalyzed reaction

Nickolay Zhadin, Robert Callender

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Laser-induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the lactate dehydrogenase-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the lactate dehydrogenase (LDH) reaction system in the presence of varying amounts of trimethylamine N-oxide (TMAO), a protein-stabilizing osmolyte, or urea, a protein-destabilizing osmolyte. Trimethylamine N-oxide (TMAO) at a concentration of 1 M strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea, the hydride transfer enthalpy is almost nullified, but the activation energy of the step is not affected significantly. TMAO increases the preference of the closed conformation of the active site loop in the LDH•NAD+• lactate complex; urea decreases it. The loop opening rate in the LDH•NADH•pyruvate complex changes its temperature dependence to inverse Arrhenius with TMAO. In this complex, urea accelerates the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius decrease in the pyruvate binding rate in the presence of TMAO offers a decrease in the fraction of the open loop, pyruvate binding competent form at higher temperatures. The pyruvate off rate is not affected by urea but decreases with TMAO. Thus, the osmolytes strongly affect the rates and thermodynamics of specific events along the LDH-catalyzed reaction: binding of substrates, loop closure, and the chemical event. Qualitatively, these results can be understood as an osmolyte-induced change in the energy landscape of the protein complexes, shifting the conformational nature of functional substates within the protein ensemble.

Original languageEnglish (US)
Pages (from-to)1582-1589
Number of pages8
JournalBiochemistry
Volume50
Issue number10
DOIs
StatePublished - Mar 15 2011

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L-Lactate Dehydrogenase
Urea
Pyruvic Acid
Hydrides
Enthalpy
Proteins
Temperature
Activation energy
Thermodynamics
NAD
Conformations
trimethyloxamine
Lactic Acid
Rate constants
Catalytic Domain
Spectrum Analysis
Lasers
Fluorescence
Spectroscopy
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effect of osmolytes on protein dynamics in the lactate dehydrogenase- catalyzed reaction. / Zhadin, Nickolay; Callender, Robert.

In: Biochemistry, Vol. 50, No. 10, 15.03.2011, p. 1582-1589.

Research output: Contribution to journalArticle

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