On the pathway of forming enzymatically productive ligand-protein complexes in Lactate Dehydrogenase
Hua Deng 1, Scott Brewer 2, Dung Vu 3, Keith Clinch 4, Robert Callender 1* and Brian R. Dyer 5
1 Albert Einstein College of Medicine
2 Los Alamos National Laboratory
3 Los Alamos National Labs
4 Industrial Research, New Zealand
5 Los Alamos National Lab
* To whom correspondence should be addressed. E-mail: call{at}aecom.yu.edu.
Submitted on January 7, 2008
Revised on February 25, 2008
Accepted on 18 March 2008
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Abstract |
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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 non-ligated 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 FTIR 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 IR 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.
Key Words:
IR, enzyme dynamics, kinetics, ligand binding, temperature jump
