RAMAN-SPECTROSCOPIC STUDIES OF THE EFFECTS OF SUBSTRATE BINDING ON COENZYMES BOUND TO LACTATE-DEHYDROGENASE

We have studied how known changes in protein structure, i.e., "loop closure" in lactate dehydrogenase brought about by the binding of substrate, affect the Raman difference spectra of both bound NADH coenzyme and other catalytically active analogues. The Raman spectrum of the NADH coenzyme in binary enzyme-NADH complex is compared to its spectrum in ternary enzyme-NADH-substrate analogue complex. As expected, we find that the protein conformational change does not modify the binding patterns of the adenosine moiety of NADH. On the other hand, there are a number of changes in the Raman spectra of the dihydronicotinamide ring of NADH and its dihydropyridine analogue, especially the vibrational modes related with the amide -NH2 of NADH and C=O of PAADH motions. On the basis of our preliminary normal mode analysis of the Raman data and other studies, we suggest that the amide C=O bond of NADH changes from a loose cisoid (to N1 nitrogen) conformation in solution to a tightly hydrogen bonded transoid conformation in LDH. Furthermore, a decrease in the number of accessible conformational states available to NADH is observed when a substrate analogue oxamate binds to the binary complex as judged by the narrowing of certain Raman bands, particularly the C4-H stretch mode of NADH. From this data, we calculate that an entropy loss corresponding to approximately 0.7 kcal/mol of free energy is associated with this constraint of NADH nicotinamide ring, and another approximately 0.7 kcal/mol is associated with the constraint of the carbonyl of pyruvate. On the basis of this, we suggest that, of the 4.2 kcal/mol decrease of the transition state barrier for hydride transfer caused by the loop closure,1 at least 1.4 kcal/mol arises from elimination of various nonproductive conformations of nicotinamide ring and pyruvate upon binding of the substrate.