Interaction of glycine zipper fragments of Aβ-peptides with neuronal nitric oxide synthase: kinetic, thermodynamic and spectrofluorimetric analysis.

Five peptide fragments [Aβ(17-21); Aβ(25-29); Aβ(29-33); Aβ(33-37); Aβ(25-37)] of the toxic Aβ(1-40(42)) amyloid peptide were shown to bind with neuronal nitric oxide synthase by means of hydrophobic-hydrophobic forces. The enzyme has a single site for the amyloid peptide binding, which resulted in a quenching of the intrinsic fluorescence of the enzyme. Binding constants determined from Stern-Volmer analysis were between 9×10(-3) and 1.8×10(-2) μM(-1). As temperature increased these binding constants increased reflecting that the interaction of the amyloid peptides with nNOS was endothermic and the quenching was dynamic. Kinetic analysis revealed a non-competitive interaction of the amyloid peptides to the enzyme with inhibitor constants of 5.1 μM for Aβ(17-21) to about 8-12 μM for the other peptides. According to the van't Hoff relationship the thermodynamic parameters, ΔH, ΔS and ΔG for the interaction of the amyloid peptides were all positive and between 41.28 and 77.86 kJ mol(-1)K(-1), 104.92 and 220.82 J mol(-1)K(-1) and 9.92 and 13.13 kJ mol(-1)K(-1), respectively. This suggested that the transition state, created by the amyloid peptide-nNOS complex and generated during the initial stages of Aβ aggregation had to, initially, overcome an activation barrier. Since the ΔG values decreased as temperature increased it not only implied a non-spontaneous interaction but that hydrophobic forces were operative during the binding. By FRET analysis the distance between the donor enzyme and the acceptor amyloid peptide was between 2.7 and 2.8 nm. As the temperature increased from 298 K through 313 K (and higher) the fraction of these tryptophan residues that became exposed increased, to approach a value of 1. There was strong support for the initial interaction being through the glycine zipper regions of Aβ(25-37).