Lactate dehydrogenase undergoes a substantial structural change to bind its substrate

Linlin Qiu, Miriam Gulotta, Robert Callender

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH·substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 ± 6 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior.

Original languageEnglish (US)
Pages (from-to)1677-1686
Number of pages10
JournalBiophysical Journal
Volume93
Issue number5
DOIs
StatePublished - Sep 2007

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L-Lactate Dehydrogenase
Temperature
Thermodynamics
NAD
Urea
Proteins
Hydrogen Bonding
Hydrogen
Spectrum Analysis
Hot Temperature
Ligands
Population
trimethyloxamine

ASJC Scopus subject areas

  • Biophysics

Cite this

Lactate dehydrogenase undergoes a substantial structural change to bind its substrate. / Qiu, Linlin; Gulotta, Miriam; Callender, Robert.

In: Biophysical Journal, Vol. 93, No. 5, 09.2007, p. 1677-1686.

Research output: Contribution to journalArticle

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