Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase

Sam Hay, Christopher Pudney, Michael J Sutcliffe, Nigel S Scrutton

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The reductive half-reaction of morphinone reductase involves a hydride transfer from enzyme-bound β-nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pKa of the reduced FMN N1 is 7.4±0.7, based on the pH-dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H-transfer as both the rate and temperature-dependence of the reaction are insensitive to changes in solution pH above and below this pKa. Further, the solvent kinetic isotope effect is ∼1.0 and both the 1° and 2° KIEs are insensitive to solution pH. The effect of the solvent’s dielectric constant is investigated and the rate of H-transfer is found to be unaffected by changes in the dielectric constant between ∼60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H-tunnelling reaction is insensitive to such changes.
Original languageEnglish
Pages (from-to)1875-1881
JournalChemPhysChem
Volume9
Issue number13
Early online date29 Jul 2008
DOIs
Publication statusPublished - 15 Sep 2008

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