Abstract
Hydride transfer is widespread in nature and has an essential role in applied research. However, the mechanisms of how this transformation occurs in living organisms remain a matter of vigorous debate. Here, we examined dihydrofolate reductase (DHFR), an enzyme that catalyzes hydride from C4′ of NADPH to C6 of 7,8-dihydrofolate (H2F). Despite many investigations of the mechanism of this reaction, the contribution of polarization of the π-bond of H2F in driving hydride transfer remains unclear. H2F was stereospecifically labeled with deuterium β to the reacting center, and β-deuterium kinetic isotope effects were measured. Our experimental results combined with analysis derived from QM/MM simulations reveal that hydride transfer is triggered by polarization at the C6 of H2F. The σ Cβ–H bonds contribute to the buildup of the cationic character during the chemical transformation, and hyperconjugation influences the formation of the transition state. Our findings provide key insights into the hydride transfer mechanism of the DHFR-catalyzed reaction, which is a target for antiproliferative drugs and a paradigmatic model in mechanistic enzymology.
Highlights
A ubiquitous event found in all living organisms, has been subjected to intense investigation with the aim of deciphering the physicochemical basis of enzyme catalysis.[1,2]
Using dihydrofolate reductase (DHFR) as a model system, we explored the role played by hyperconjugation in driving hydride transfer
Primary kinetic isotope effect (KIE) arise when atoms directly involved in the chemical transformation are replaced by their heavy counterparts.[6,19,21−23] Primary KIE measurements for NADPH(D) and heavy-atom (15N, 13C) isotope labeling of the primary reacting centers have generated evidence in support of a stepwise mechanism for DHFR from E. coli (EcDHFR).[21,22,24,25]
Summary
A ubiquitous event found in all living organisms, has been subjected to intense investigation with the aim of deciphering the physicochemical basis of enzyme catalysis.[1,2] Despite a wealth of studies,[3−8] a model that comprehensively illustrates the unparalleled catalytic power of enzymes is still lacking. The kinetic isotope effect (KIE) is a powerful tool to investigate enzyme mechanisms.[8,9,18−20] During DHFR catalysis, hydride transfer to C6 and protonation of the N5 of H2F occur (see Figure 1). Both N5 and C6 of H2F change from sp[2] to sp[3] hybridization, while C4′ of NADPH alternates from sp[3] to sp[2]. Primary KIEs arise when atoms directly involved in the chemical transformation are replaced by their heavy counterparts.[6,19,21−23] Primary KIE measurements for NADPH(D) and heavy-atom (15N, 13C) isotope labeling of the primary reacting centers have generated evidence in support of a stepwise mechanism for DHFR from E. coli (EcDHFR).[21,22,24,25] Secondary α-deuterium KIEs (α-KIEs) arising from the rehybridization of C4′ of NADPH provide atomistic insights into local environmental changes during the chemical transformation, as isotopic substitution influences the rehybridization process of the primary atoms, which is reflected as a change in reaction rate.[23,26−28] despite EcDHFR being one of the most studied enzymes, the role of C6 rehybridization in H2F has never been investigated in Received: July 12, 2019 Revised: September 20, 2019 Published: September 23, 2019
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