Abstract
Understanding the mechanisms of enzymatic hydride transfer with nicotinamide coenzyme biomimetics (NCBs) is critical to enhancing the performance of nicotinamide coenzyme-dependent biocatalysts. Here the temperature dependence of kinetic isotope effects (KIEs) for hydride transfer between "better than nature" NCBs and several ene reductase biocatalysts is used to indicate transfer by quantum mechanical tunneling. A strong correlation between rate constants and temperature dependence of the KIE (ΔΔH(⧧)) for H/D transfer implies that faster reactions with NCBs are associated with enhanced donor-acceptor distance sampling. Our analysis provides the first mechanistic insight into how NCBs can outperform their natural counterparts and emphasizes the need to optimize donor-acceptor distance sampling to obtain high catalytic performance from H-transfer enzymes.
Highlights
The search for synthetic biomimetics that can replace natural nicotinamide coenzymes has been driven by the instability and expense of NAD(P)H
nicotinamide coenzyme biomimetics (NCBs) support biocatalysis with many ene reductases (ERs),[12,14] which belong to the Old Yellow Enzyme (OYE) family (EC 1.3.1.31)
We have reported crystal structures of selected ER-NCB complexes, a comprehensive analysis of reactions catalyzed by 12 ERs with 5 synthetic NCBs, and coenzyme analogue recycling to demonstrate the overall effectiveness of NCBs in ER-catalyzed biotransformations.[14]
Summary
Communication constants vary by 200-fold at 25 °C, from 2 s−1 (PETNR:NADH) to 434 s−1 (TOYE:mBu). The reduction potentials of NADH and NADPH are not significantly different, yet the reactions of all three ERs with NADPH are 10−20-fold faster than with NADH In both PETNR and XenA, NADPH reacts as fast as or faster than mNH2, despite the likely larger driving force of the mNH2 reaction.[40] Modest changes to the reaction driving force are not expected to alter the magnitude of the KIE,[31,41] so the kinetic differences observed between natural and biomimetic coenzymes do not appear to solely arise through differing reaction driving forces.
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