Diphtheria toxin catalyzed hydrolysis of NAD(+): Molecular dynamics study of enzyme-bound substrate, transition state, and inhibitor

The mechanism of the diphtheria toxin-catalyzed hydrolysis of NAD(+) was in vestigated by quantum chemical calculations and molecular dynamics simulati ons. Several effects that could explain the 6000-fold rate acceleration (De lta DeltaG(double dagger) similar to 5 kcal/mol) by the enzyme were conside red. First, the carboxamide arm of the enzyme-bound NAD(+) adopts a trans c onformation while the most stable conformation is cis. The most stable conf ormation for the nicotinamide product has the amide carbonyl trans. The act ivation energy for the cleavage of the ribosidic bond is reduced by 2 kcal/ mol due to the relaxation of this ground state conformational stress in the transition state. Second, molecular dynamics simulations to the nanosecond time range revealed that the carboxylate of Glu148 forms a hydrogen bond t o the substrate's 2' hydroxyl group in E.S (similar to 17% of the time) and E.TS (similar to 57% of the time) complexes. This interaction is not seen in crystal structures. The ApUp inhibitor is held more tightly by the enzym e than the transition state and the substrate. Analysis of correlated motio ns reveals differences in the pattern of anticorrelated motions for protein backbone atoms when the transition state occupies the active site as compa red to the E.NAD(+) complex.