Kinetics of enzyme-coenzyme interactions by NMR spectroscopy.
The kinetics for the binding of coenzymes to H4 and M4 lactate dehydrogenase from chicken were investigated by nuclear magnetic resonance spectroscopy. With detailed computer analysis, some kinetic parameters were extracted from the chemical shifts and the linewidth of the observed coenzyme resonances at various enzyme/coenzyme ratios and temperatures. The results of the analysis indicated that the dissociation rates of coenzymes from the enzyme/coenzyme complexes are slower with the H4 isozyme than those involving the M4 isozyme. The lifetimes for the NAD+-enzyme complexes are on the order of 1 msec while those for the NADH-enzyme complexes are on the order of 10 ms (at room temperature). Much shorter transverse relaxation times of the coenzyme resonances were observed in NADH-enzyme complexes than those in the NAD+-enzyme complexes. The calculated kinetic constants are in good agreement with the previous studies by stopped-flow and temperature jump methods. A generalized NMR kinetic treatment for the binding of small molecules to a macromolecule is presented.
D000818:Animals; D002645:Chickens; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D009243:NAD; D010084:Oxidation-Reduction; D011485:Protein Binding
D007527:Isoenzymes; D009243:NAD; D007770:L-Lactate Dehydrogenase