Anticorrelated motions as a driving force in enzyme catalysis: the dehydrogenase reaction

Abstract

Molecular dynamics and cross-correlation analysis of the horse liver alcohol dehydrogenase HLADH.NAD(+).PhCH(2)O(-) complex has established anticorrelated motions between the NAD(+)-binding domain and other portions of the enzyme. Four pairs of anticorrelated interactions are (i and ii) cofactor-binding domain: C(alpha) of V292 and the CG1 of V203 with C7 of PhCH(2)O(-); (iii) cofactor-binding domain: amide carbonyl oxygen of I318 with amide N of H67; and (iv) cofactor domain: C(alpha) of T178 with carbonyl oxygen of L141. The average distances between pairs are 9.2 A for i, 8.2 A for ii, 14.7 A for iii, and 18.2 A for iv. The motions of i and ii are most important in the approximately 0.5 A pushing of C4 of NAD(+) toward C7 of PhCH(2)O(-) to form push near-attack conformer (NACs). The motions of iv are less so, and those of iii are not important. Seventy-five quantum mechanics/molecular mechanics calculations of the energies of reaction were carried out without structural restrictions from different stages of the molecular dynamics trajectory. Of the 71 conformations, the 29 fulfilling NAC criteria were associated with the lowest energies of activation. Thus, anticorrelated motions from the NAD(+)-binding domain by way of the neighboring V292 and V203 have a pushing motion, which moves the C4 of NAD(+) toward the H-C7 of the substrate. Longer-range anticorrelated motions involving the cofactor-binding domain have no or very little influence on NAC formation.

Fig. 1.

Anticorrelated motions mapped onto the monomer structure. NAD⁺ and PhCH₂O^– are shown by pink and cyan dotted surfaces, respectively. The regions of the anticorrelated motions are assigned and labeled.

Fig. 2.

The geometric placement of chosen anticorrelated pairs in the HLADH·NAD⁺·PhCH₂O^– complex. The view angle of the stereo structure shown is a flip-over from Fig. 1.

Fig. 3.

Distance histograms for four anticorrelated pairs. Histograms of the distances between C7 of PhCH₂O^– and NC4 of NAD⁺ plotted vs. the distances between C^α of V292 and C7 of PhCH₂O^– (A), the distances between CG1 of V203 and C7 of PhCH₂O^– (B), the distances between the carbonyl oxygen of I318 and the nitrogen of H67 (C), and the distances between the carbonyl oxygen of L141 and the C^α of T178 (D). Slopes of the dashed lines are calculated directly from x–y coordinates. In three dimensions, the motion of L141 toward T178 is seen to create three different protein conformations without change in the distances separating reactants.

Fig. 4.

The histogram plot of the distances C7 of PhCH₂O^–···NC4 of NAD⁺ vs. the angles C7–H···NC4; reactions exhibiting the lowest values of energy-barrier heights are projected as red dots. The higher values of energy-barrier heights are represented as green dots. The most reactive conformations are located in the area of the distribution of conformations by the inserted box, which defines (5) the NACs.

Fig. 5.

The lowest values of energy-barrier heights are projected as red dots on the distance histograms of Fig. 3. Data points in A–C are duplicates of those shown in Fig. 3 A–C with the addition of the recognition of the conformers with the lowest energies of reaction. Reactions exhibiting the lowest values of energy-barrier heights are projected as red dots on the histograms of the distances between C7 of PhCH₂O^– and NC4 of NAD⁺ plotted vs. the distances between C^α of V292 and C7 of PhCH₂O^– (A), the distances between CG1 of V203 and C7 of PhCH₂O^– (B), and the distances between the carbonyl oxygen of I318 and the nitrogen of H67 (C).