On the pathway of forming enzymatically productive ligand-protein complexes in lactate dehydrogenase.
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.
Deng
Hua
H
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Brewer
Scott
S
Vu
Dung M
DM
Clinch
Keith
K
Callender
Robert
R
Dyer
R Brian
RB
D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000648:ultrastructure*; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*
D008024:Ligands; D007770:L-Lactate Dehydrogenase