Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase.

Appleman, James R.; Beard, William A.; Delcamp, Tavner J.; Prendergast, Neal J.; Freisheim, James H.; Blakley, Raymond L.

doi:10.1016/s0021-9258(19)39864-3

Cited by 68 publications

(98 citation statements)

References 38 publications

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“…Finally, the product THF is released from the active site in the rate-limiting step at pH 7 and the enzyme returns to the closed conformation and another reduced cofactor enters the active site. 16 A number of DHFRs have been shown to follow similar catalytic cycles to EcDHFR, including DHFR from humans 32 as well as those from bacteria including Lactobacillus casei (LcDHFR) 33 and the psychrophile Moritella profunda (MpDHFR). 34 In all these cases, and for DHFR from the thermophilic bacterium Geobacillus stearothermophilus (BsDHFR), 35,36 the steady state turnover at pH 7 is limited by a physical step rather than the actual chemical step of hydride transfer.…”

Section: Introductionmentioning

confidence: 99%

“…[39][40][41] The kinetic isotope effects (KIE) on the DHFR-catalysed reaction have been measured chiefly by two experimental techniques. For many DHFRs, the turnover number k cat at pH 7 reports mainly on product release 14,[32][33][34] and transient kinetic techniques such as stopped flow must be used to extract information about the chemical step of the catalytic cycle. These have the advantage that they can be performed under conditions where hydride transfer makes little or no contribution to k cat .…”

Section: Introductionmentioning

confidence: 99%

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Protein motions and dynamic effects in enzyme catalysis

Luk

Loveridge

Allemann

2015

Phys. Chem. Chem. Phys.

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The role of protein motions in promoting the chemical step of enzyme catalysed reactions remains a subject of considerable debate. Here, a unified view of the role of protein dynamics in dihydrofolate reductase catalysis is described. Recently the role of such motions has been investigated by characterising the biophysical properties of isotopically substituted enzymes through a combination of experimental and computational analyses. Together with previous work, these results suggest that dynamic coupling to the chemical coordinate is detrimental to catalysis and may have been selected against during DHFR evolution. The full catalytic power of Nature's catalysts appears to depend on finely tuning protein motions in each step of the catalytic cycle.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Protein motions and dynamic effects in enzyme catalysis

Luk

Loveridge

Allemann

2015

Phys. Chem. Chem. Phys.

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“…A notable difference between the cellular environments of prokaryotic and eukaryotic cells is the intracellular pool of NADP + and NADPH . Since the catalytic turnover of both prokaryotic and eukaryotic DHFRs is limited by product release, , there was evolutionary pressure to respond to changing cellular environments while maintaining an efficient rate of enzyme-catalyzed hydride transfer. The PCE that introduced the polyproline sequence restricts the motion of the M20 loop that undergoes dramatic conformational changes during the turnover of ecDHFR .…”

Section: Introductionmentioning

confidence: 99%

Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase

Lin

Kohen

et al. 2021

Biochemistry

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Evolution of dihydrofolate reductase (DHFR) has been studied using the enzyme from Escherichia coli DHFR (ecDHFR) as a model, but less studies have used the enzyme from Homo sapiens DHFR (hsDHFR). Each enzyme maintains a short and narrow distribution of hydride donor-acceptor distances (DAD) at the tunneling ready state (TRS). Evolution of the enzyme was previously studied in ecDHFR where three key sites were identified as important to the catalyzed reaction. The corresponding sites in hsDHFR are F28, 62-PEKN, and 26-PPLR. Each of these sites was studied here through the creation of mutant variants of the enzyme and measurements of the temperature dependence of the intrinsic kinetic isotope effects (KIEs) on the reaction. F28 is mutated first to M (F28M) and then to the L of the bacterial enzyme (F28L). The KIEs of the F28M variant are larger and more temperature-dependent than wild-type (WT), suggesting a broader and longer average DAD at the TRS. To more fully mimic ecDHFR, we also study a triple mutant of the human enzyme (F32L-PP26N-PEKN62G). Remarkably, the intrinsic KIEs, while larger in magnitude, are temperature-independent like the WT enzymes. We also construct deletion mutations of hsDHFR removing both the 62-PEKN and 26-PPLR sequences. The results mirror those described previously for insertion mutants of ecDHFR. Taken together, these results suggest a balancing act during DHFR evolution between achieving an optimal TRS for hydride transfer and preventing product inhibition arising from the different intercellular pools of NADPH and NADP + in prokaryotic and eukaryotic cells.

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“…[1a,d,2] Measurements of kinetic isotope effects (KIEs) can shed light on how changes in protein dynamics from isotopic substitution effect enzyme catalysis. KIEs obtained when isotopes ( 1 H, 12 C and 14 N) of a substrate or an enzyme are replaced with their heavy counterparts ( 2 H, 13 C and 15 N) report on bonding and geometry differences between reactants and the transition state. [3] However, for enzymatic reactions, the measurement of KIEs is often difficult and the real KIE value can become masked when the elementary step of the reaction is fast or the observed rate constant reflects multiple kinetic steps.…”

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confidence: 99%

Cryo‐kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase

2021

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Effects of isotopic substitution on the rate constants of human dihydrofolate reductase (HsDHFR), an important target for anti‐cancer drugs, have not previously been characterized due to its complex fast kinetics. Here, we report the results of cryo‐measurements of the kinetics of the HsDHFR catalyzed reaction and the effects of protein motion on catalysis. Isotopic enzyme labeling revealed an enzyme KIE (kHLE/kHHE) close to unity above 0 °C; however, the enzyme KIE was increased to 1.72±0.15 at −20 °C, indicating that the coupling of protein motions to the chemical step is minimized under optimal conditions but enhanced at non‐physiological temperatures. The presented cryogenic approach provides an opportunity to probe the kinetics of mammalian DHFRs, thereby laying the foundation for characterizing their transition state structure.

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Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase.

Cited by 68 publications

References 38 publications

Protein motions and dynamic effects in enzyme catalysis

Protein motions and dynamic effects in enzyme catalysis

Evolution of Optimized Hydride Transfer Reaction and Overall Enzyme Turnover in Human Dihydrofolate Reductase

Cryo‐kinetics Reveal Dynamic Effects on the Chemistry of Human Dihydrofolate Reductase

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