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Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli.
- Source :
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Journal of Biological Chemistry . 12/2/2016, Vol. 291 Issue 49, p25411-25426. 16p. - Publication Year :
- 2016
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Abstract
- 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, Kd = 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 kcat (=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. Amodel of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00219258
- Volume :
- 291
- Issue :
- 49
- Database :
- Academic Search Index
- Journal :
- Journal of Biological Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 119961416
- Full Text :
- https://doi.org/10.1074/jbc.M116.747014