EVALUATION OF THE FACTORS INFLUENCING REACTIVITY AND STEREOSPECIFICITY IN NAD(P)H DEPENDENT DEHYDROGENASE ENZYMES

Calculations of AM1 potential energies for conformations of N-(1,beta-ribosyl)-1,4-dihydronicotinamide (3H) and N-(1,beta-ribosyl)nicotinamide (3), as defined by the nicotinamide ring deformation angles alpha(C) and alpha(N) and the torsion angles X(n) and X(am), have been carried out. X(n) is the angle of rotation of the nicotinamide ring relative to the ribose ring and the conformers are designated as syn (nicotinamide -CONH2 and ribose ring O are, close) and anti (nicotinamide -CONH2 and ribose ring O are distant). The angles alpha(C) and alpha(N) reflect the degree of bending of nicotinamide N and C4 out of the plane with C2, C3, C5, and C6 to provide quasi-boat and quasi-half-chair conformations. With the 1,4-dihydronicotinamide ring in a quasi-boat geometry, the relationship of the anti-bonding orbital on the ribose ether O and the unshared electron pair on the nicotinamide ring N is described as periplanar or antiperiplanar. The quasi-boat conformations may then be described as anti antiperiplanar (1a), syn antiperiplanar (1b), anti periplanar (1c), and syn periplanar (1d). The AM1 potential energies expended in the deformation of the 1,4-dihydropyridine ring to quasi-boat or quasi-half-chair conformations (with alpha(C) + alpha(N) less-than-or-equal-to 20-degrees and alpha(C) greater-than-or-equal-to alpha(N)) arc less than the gain in the accompanying stabilization of the transition state in the reduction of a carbonyl function by transfer of the pseudoaxial hydrogen at C4. With alpha(C) = 15-degrees and alpha(N) = 5-degrees, the potential energies of 3 as a function of X(n) is raised relative to its global minimum by an average value of 16 kcal/mol. To bend the flat 1,4-dihydronicotinamide of 3H such that alpha(C) = 15-degrees and alpha(N) = 5-degrees costs 1.8 kcal/mol, while the activation enthalpy is decreased by 6 kcal/mol compared with the flat nicotinamide. In the bent conformation, the initial state {3H + H2C=O...HImH+} and immediate product state {3 + H3COH...ImH} potential energies are not greatly different. Energetically, there is little difference as to whether H(R) or H(S) are the transferred entities at the pseudoaxial positions. From structures deposited in the Brookhaven database, X(n) = -87-degrees to -140-degrees (conformation 1a) for A-specific and X(n) = 45-degrees to 66-degrees (conformation 1b) for B-specific dehydrogenases. The mismatch in the preferred values of X(n) for unbound (AM1) and enzyme bound (Brookhaven database) reduced and oxidized cofactors has little influence on the equilitrium constants for cofactor binding which favors the reduced form. CHARM(m) molecular dynamics simulations show the deformation of the alpha(C) + alpha(N) of 3H to A- and B-sides is isoenergetic when 3H is not enzyme bound. The same experiments with dogfish muscle lactate dehydrogenase show the motions of the NAD+ and NADH torsional angles X(n) and X(am), as well as the puckering angle alpha(C) for C4, are quite flexible as predicted by AM1 calculations. Molecular dynamics simulations with lobster D-glyceraldehyde 3-phosphate dehydrogenase, L. casei dihydrofolate reductase, and porcine heart malate dehydrogenase also show the puckering angle alpha(C) for C4 of NADH to be quite flexible. With the various dehydrogenases the puckering motion of NADH is anisotropic, such that the C4 predominantly bends toward the substrate binding site. For each enzyme, steric hindrance by hydrophobic residues on the distal face of the cofactor are responsible for this anisotropic movement. In the dogfish lactate dehydrogenase pyruvate-NADH complex, the transferable pseudoaxial H(R) at C4 comes within van der Waals contact with the carbonyl carbon of the substrate. Further, dynamics calculations indicate that the protonated imidazole group of HIS 193 is required not only as a general-acid catalyst but also for the correct alignment of pyruvate. The important catalytic features of the dogfish lactate dehydrogenase were examined by AM1 calculations using the X-ray coordinates for the enzyme around the active site and by molecular dynamics calculations using the entire enzyme. These features are the following: (i) in the ground state, approach of all reactants to within van der Waals radii; (ii) in the transition state, proton transfer from imidazolium cation (ImH-2+) of HIS 193 to substrate carbonyl oxygen is almost complete while hydrogen transfer from 1,4-dihydronicotinamide to carbonyl carbon is about midway. The late transition state for proton transfer from ImH-2+ is facilitated by the repulsive positive charges of ImH-2+ and the arginine guanidinium cation substituent of ARG 106. It is proposed that the major driving forces in the catalysis are the steric compression in the ground state, the lateness of proton transfer which provides a large partial positive charge on the carbonyl carbon, and the preequilibrium puckering of the 1,4-dihydronicotinamide ring to quasi-boat conformation with transfer of the pseudoaxial hydrogen. The favoring of A- or B-side hydrogen transfer is determined by shielding of one side of the 1,4-dihydronicotinamide ring, which prevents access of substrate to and dynamic deformation at the unblocked side. An earlier proposal that dehydrogenases evolved in such a manner to level their dynamics by having NADH assume the weaker reductant anti antiperiplanar conformation (1a) in H(R) transfer (A-side) and the stronger reductant syn antiperiplanar conformation (1b) in H(S) transfer (B-side) cannot be correct on the basis of the AM1 potential energies of these conformations (i.e., 1a and 1b should be comparable reducing agents). In L. casei dihydrofolate reductase, the weakly polar interactions of oxygen functions of THR 45, ILE 13, and ALA 97 with the hydrogens of C2-H, C4-H, and C6-H of NADPH do not represent hydrogen bonds previously thought to be important in the activation of the cofactor. In the dehydrogenases in general, the amino acid oxygen functionalities which point inwards toward the nicotinamide ring are involved in hydrogen bonds to water molecules in the apoenzyme thereby keeping the cofactor binding site open.