On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase

Hua Deng, Scott Brewer, Dung M. Vu, Keith Clinch, Robert Callender, R. Brian Dyery

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We have carried out a series of studies on the binding of a substrate mimic to the enzyme lactate dehydrogenase (LDH) using advanced kinetic approaches, which begin to provide a molecular picture of the dynamics of ligand binding for this protein. Binding proceeds via a binding-competent subpopulation of the nonligated form of the protein (the LDH/NADH binary complex) to form a protein-ligand encounter complex. The work here describes the collapse of the encounter complex to form the catalytically competent Michaelis complex. Isotope-edited static Fourier transform infrared studies on the bound oxamate protein complex reveal two kinds of oxamate environments: 1), a major populated structure wherein all significant hydrogen-bonding patterns are formed at the active site between protein and bound ligand necessary for the catalytically productive Michaelis complex and 2), a minor structure in a configuration of the active site that is unfavorable to carry out catalyzed chemistry. This latter structure likely simulates a dead-end complex in the reaction mixture. Temperature jump isotope-edited transient infrared studies on the binding of oxamate with LDH/NADH suggest that the evolution of the encounter complex between LDH/NADH and oxamate collapses via a branched reaction pathway to form the major and minor bound species. The production of the catalytically competent protein-substrate complex has strong similarities to kinetic pathways found in two-state protein folding processes. Once the encounter complex is formed between LDH/NADH and substrate, the ternary protein-ligand complex appears to "fold" to form a compact productive complex in an all or nothing like fashion with all the important molecular interactions coming together at the same time.

Original languageEnglish (US)
Pages (from-to)804-813
Number of pages10
JournalBiophysical Journal
Volume95
Issue number2
DOIs
StatePublished - Jul 15 2008

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L-Lactate Dehydrogenase
NAD
Ligands
Proteins
Isotopes
Catalytic Domain
Ternary Complex Factors
Protein Folding
Fourier Analysis
Molecular Dynamics Simulation
Hydrogen Bonding
Carrier Proteins
Temperature
Enzymes

ASJC Scopus subject areas

  • Biophysics

Cite this

On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase. / Deng, Hua; Brewer, Scott; Vu, Dung M.; Clinch, Keith; Callender, Robert; Dyery, R. Brian.

In: Biophysical Journal, Vol. 95, No. 2, 15.07.2008, p. 804-813.

Research output: Contribution to journalArticle

Deng, Hua ; Brewer, Scott ; Vu, Dung M. ; Clinch, Keith ; Callender, Robert ; Dyery, R. Brian. / On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase. In: Biophysical Journal. 2008 ; Vol. 95, No. 2. pp. 804-813.
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