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On the Pathway of Forming Enzymatically Productive Ligand-Protein Complexes in Lactate Dehydrogenase

Hua Deng ^*, Scott Brewer , Dung M. Vu , Keith Clinch , Robert Callender ^* and R. Brian Dyer 

^* Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, 10461; Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545; and Industrial Research, Lower Hutt, New Zealand

Correspondence: Address reprint request to Dr. Robert Callender, Tel.: 718-430-3024; Fax: 718-430-8565; E-mail: call{at}aecom.yu.edu; or R. B. Dyer, Tel.: 505-667-4194; Fax: 505-667-0851; E-mail: bdyer{at}lanl.gov.

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.

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