[Related articles/posters: None: require molecules]

Gideon Fleminger⁺, Etty Kochavi* and Akiva Bar-Nun*, ⁺Department of Molecular Microbiology and Biotechnology and *Department of Geophysics and Planetary Sciences, Tel Aviv University, Tel Aviv, Israel, 69978

Enzymatic reactions involve the association of specific amino acid residues in the enzyme active site with certain groups on the substrate molecule, via a series of subtle, non-covalent interactions. We suggest that this type of specific recognition, under certain conditions, can be utilized for substrate-induced synthesis of catalytic oligopeptides. Incubation of a given substrate with a mixture of amino acids (and possibly with certain metal ions) may cause their assembly into an "active-site-like" pocket. Upon addition of a condensing agent, a (metallo)oligopeptide should be formed, which interacts with the substrate in a specific manner. Under certain conditions, the newly-formed molecule should act on the substrate as an enzyme. To examine this hypothesis experimentally, we used the substrate o-nitrophenol--D-galactopyranoside (ONPG) as a molecular template to assemble certain amino acids and to synthesize a specific catalyst, capable of cleaving the same substrate. This was achieved by incubation of ONPG with a mixture of free amino acids, FeSO₄ and a condensing agent (dicyandiamide) at elevated temperatures. The reaction rate (d[product]/d²t) increased linearly with time, indicating an acceleration regime, where the substrate generates the formation of a catalyst. This catalyst was purified by RP-HPLC and identified as Cys₂-Fe⁺² by a series of structural and chemical analyses. It was shown to catalyze the hydrolysis of ONPG, used to assist its own formation. Based on specificity studies, kinetic data and molecular imaging, a model for the structure and mode of action of this catalyst is proposed.

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