Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride–proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.

show abstract

“…9 Experimental studies with heavy ecDHFR support the absence of PPVs as predicted by the TPS simulations. 16–19 …”

Section: Introductionmentioning

confidence: 99%

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase

2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…54 The main error in ref 54 is that instead of using the reciprocal of the measured KIE used in the Northrop equation (Eq 1 here and S12 in ref 54), they derived normal KIEs, which lead to the commitments for hydrogen and deuterium (Eqs S2–S5 in ref 54), but not for tritium. Consequently, their derivations did not lead to the necessary isolation of the commitment for tritium (C fT ) needed in the Northrop procedure with H/T and D/T measurements.…”

mentioning

confidence: 99%

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

et al. 2016

View full text Add to dashboard Cite

Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general.

show abstract

“…The correct procedure described above (Eqs. 3-14) can be used for the complex kinetic scheme used in ref (Z. Wang et al, 2016) to calculate CT directly, without the use of any further assumptions.…”

Section: The Northrop Methodsmentioning

confidence: 99%

Examinations of the Chemical Step in Enzyme Catalysis

Singh

Islam

Kohen

2016

Methods in Enzymology

View full text Add to dashboard Cite

Advances in computational and experimental methods in enzymology have aided comprehension of enzyme-catalyzed chemical reactions. The main difficulty in comparing computational findings to rate measurements is that the first examines a single energy barrier while the second frequently reflects a combination of many microscopic barriers. We present here intrinsic kinetic isotope effects and their temperature dependence as a useful experimental probe of a single chemical step in a complex kinetic cascade. Computational predictions are tested by this method for two model enzymes: dihydrofolate reductase (DHFR) and thymidylate synthase (TSase). The description highlights the significance of collaboration between experimentalists and theoreticians to develop a better understanding of enzyme-catalyzed chemical conversions.

show abstract

Hydride Transfer in DHFR by Transition Path Sampling, Kinetic Isotope Effects, and Heavy Enzyme Studies

Cited by 33 publications

References 54 publications

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

Examinations of the Chemical Step in Enzyme Catalysis

Contact Info

Product

Resources

About