Etiology of Alzheimer's disease: kinetic, thermodynamic and fluorimetric analyses of interactions of pseudo Aβ-peptides with neuronal nitric oxide synthase.
Aggregated β-amyloid deposit is a hallmark in the neuropathology of Alzheimer's disease but their mechanism of formation still remains unresolved. Previously we reported that a normal pentapeptide Aβ(17-21) and glycine zipper peptide Aβ(29-33) strongly inhibited nitric oxide synthase and rapidly initiated fibrillogenesis. Critical amino acids within these fragments were not identified. We now report on the interaction of four pseudo-peptides with nNOS - two peptides with a reversed amino acid sequence [Aβ(17-21r); Aβ(29-33r)] and two peptides with Phe19, Phe20 and Ile31, Ile32 substituted with polar glutamic acid [Aβ(17-21p); Aβ(29-33p)]. It was shown that while the inhibitor constants (Ki) increased 2-3 fold for each of the pseudo-peptides when compared with the normal peptides the dissociation constant Kd increased between 20 and 50 fold. Stern-Volmer fluorescence quenching constants (K(SV)) for Aβ(17-21p) and Aβ(29-33p) were 7.2×10(-3) and 6.1×10(-3) μM(-1) respectively at 298 K some 2-3 fold lower than the corresponding Aβ(17-21r); Aβ(29-33r). With temperature increase there was an increase in K(SV) and Kd, suggesting a dynamic quenching mechanism. Thermodynamic parameters, ΔH, ΔS and ΔG were all positive indicating endothermic, non-spontaneous, hydrophobic-hydrophobic associations of the pseudo-peptides with the enzyme. By FRET analysis the efficiency of fluorescence transfer between enzyme tryptophans and the pseudo-peptides was 90% (compared to 97% for the natural substrate). The distance the tryptophans moved after interaction with Aβ(17-21r) and Aβ(17-21p) was 10% greater, while for Aβ(29-33r) and Aβ(29-33p) it was 20-25% greater, than with the normal peptides; the fluorescence intensity was 20-75% higher. This increase in distance, fluorescent intensity and transfer efficiency illustrate an increase in interaction energy for the pseudo-peptides with nNOS lending support for the strategic position of the Phe19, Phe20, Ile31 and Ile32 in the original peptides not only for inhibition of the nNOS but for initiation of fibrillogenesis.