Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli.

The enzyme QueF catalyzes a four-electron reduction of a nitrile group into an amine, the only reaction of this kind known in biology. In nature, QueF converts 7-cyano-7-deazaguanine (preQ₀) into 7-aminomethyl-7-deazaguanine (preQ₁) for the biosynthesis of the tRNA-inserted nucleoside queuosine. The proposed QueF mechanism involves a covalent thioimide adduct between preQ₀ and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ₁ in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ₀ in a strongly exothermic process (ΔH = -80.3 kJ/mol; -TΔS = 37.9 kJ/mol, K_d = 39 nm) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ₀ reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4-7-fold more slowly than trapping of the enzyme-bound preQ₀ as covalent thioimide (1.63 s^-1) and are thus mainly rate-limiting for the enzyme's k_cat (=0.12 s^-1). Kinetic studies combined with simulation reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ₁ 7-Formyl-7-deazaguanine, a carbonyl analogue of the imine intermediate, was synthesized chemically and is shown to be recognized by QueF as weak ligand for binding (ΔH = -2.3 kJ/mol; -TΔS = -19.5 kJ/mol) but not as substrate for reduction or oxidation. A model of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data.

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