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.