Chicken avidin exhibits pseudo-catalytic properties - Biochemical, structural, and electrostatic consequences

Avidin and its bacterial analogue streptavidin exhibit similarly high affin ities toward the vitamin biotin. The extremely high affinity of these two p roteins has been utilized as a powerful tool in many biotechnological appli cations. Although avidin and streptavidin have similar tertiary and quatern ary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, wh ereas streptavidin protects this reaction even under extreme alkaline condi tions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin) . The three-dimensional crystal structures of avidin (2 Angstrom) and strep tavidin (2.4 Angstrom) complexed with the amide analogue, biotinyl p-nitroa nilide, as a model for the p-nitrophenyl ester, revealed structural insight s into the factors that enhance or protect the hydrolysis reaction. The dat a demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine III and the nit ro group of the substrate. The open conformation of the loop serves to expo se the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdra wing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analog ous interactions with the substrate were consequently absent. The informati on derived from these structures may provide insight into the action of art ificial protein catalysts and the evolution of catalytic sites in general.