Kinetic Isotope Effects in Enzymology

Klinman, Judith P.

doi:10.1002/9780470122914.ch7

Cited by 32 publications

(20 citation statements)

References 180 publications

(96 reference statements)

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“…Importantly, for multidimensional H-tunneling models, QM wave function overlap is assumed to be an instantaneous event, with the slower heavy-atom motions controlling the barrier crossing. This feature was initially counterintuitive and contrary to earlier treatises on KIEs, in which researchers concluded that for KIEs to be measurable, all steps (including protein conformation changes) would need to be rapid in relation to the H-transfer (17, 67). The separation of timescales for protein motions and hydrogenic wave function overlap that results from Equation 4 alters the underlying timescales, such that hydrogen transfer by wave function overlap is always the fastest step.…”

Section: Protein Dynamics Linked To Hydrogen Tunneling Efficiencymentioning

confidence: 83%

“…Modern enzymology has relied heavily on the use of isotope effects to determine the nature of rate-limiting steps and, later, TS structure (17–19). The theory of isotope effects developed after World War II primarily focused on a semiclassical approximation that included changes in force constants ( F ) between the ground state and TS together with an impact of the reduced mass (µ) at the reacting bond on the frequency (ν) of the vibrating/reacting bond (Equation 1) (20):

ν = 1 / 2 π \sqrt{F / μ} .

Beginning in the 1980s, investigators noted experimental observations that could not be explained in these seemingly simple terms and introduced the possibility of quantum mechanical (QM) tunneling.…”

Section: Empirical Deviations and Resulting Theoretical Models For Tumentioning

confidence: 99%

See 1 more Smart Citation

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis

Klinman

Kohen

2013

Annu. Rev. Biochem.

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307

474

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The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C–H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

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Section: Protein Dynamics Linked To Hydrogen Tunneling Efficiencymentioning

confidence: 83%

ν = 1 / 2 π \sqrt{F / μ} .

Section: Empirical Deviations and Resulting Theoretical Models For Tumentioning

confidence: 99%

Hydrogen Tunneling Links Protein Dynamics to Enzyme Catalysis

Klinman

Kohen

2013

Annu. Rev. Biochem.

Self Cite

307

474

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“…Kinetic isotope effects (KIEs) are a useful experimental probe of nonclassical behaviour in H-transfer reactions, [26] with values of primary KIEs inflated above the semiclassical limits of k H /k D % 7 at 298 K seen as an indication of tunnelling. [27] Note that the kinetic complexity of the reaction mechanism may reduce this value.…”

Section: A C H T U N G T R E N N U N G (Madh)/thr172ba C H T U N G T mentioning

confidence: 99%

Analysis of Classical and Quantum Paths for Deprotonation of Methylamine by Methylamine Dehydrogenase

et al. 2007

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The hydrogen-transfer reaction catalysed by methylamine dehydrogenase (MADH) with methylamine (MA) as substrate is a good model system for studies of proton tunnelling in enzyme reactions--an area of great current interest--for which atomistic simulations will be vital. Here, we present a detailed analysis of the key deprotonation step of the MADH/MA reaction and compare the results with experimental observations. Moreover, we compare this reaction with the related aromatic amine dehydrogenase (AADH) reaction with tryptamine, recently studied by us, and identify possible causes for the differences observed in the measured kinetic isotope effects (KIEs) of the two systems. We have used combined quantum mechanics/molecular mechanics (QM/MM) techniques in molecular dynamics simulations and variational transition state theory with multidimensional tunnelling calculations averaged over an ensemble of paths. The results reveal important mechanistic complexity. We calculate activation barriers and KIEs for the two possible proton transfers identified-to either of the carboxylate oxygen atoms of the catalytic base (Asp428beta)-and analyse the contributions of quantum effects. The activation barriers and tunnelling contributions for the two possible proton transfers are similar and lead to a phenomenological activation free energy of 16.5+/-0.9 kcal mol(-1) for transfer to either oxygen (PM3-CHARMM calculations applying PM3-SRP specific reaction parameters), in good agreement with the experimental value of 14.4 kcal mol(-1). In contrast, for the AADH system, transfer to the equivalent OD1 was found to be preferred. The structures of the enzyme complexes during reaction are analysed in detail. The hydrogen bond of Thr474beta(MADH)/Thr172beta(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471beta(MADH)/Phe169beta(AADH) are identified as important factors in determining the preferred oxygen acceptor. The protein environment has a significant effect on the reaction energetics and hence on tunnelling contributions and KIEs. These environmental effects, and the related clearly different preferences for the two carboxylate oxygen atoms (with different KIEs) in MADH/MA and AADH/tryptamine, are possible causes of the differences observed in the KIEs between these two important enzyme reactions.

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“…An isotope effect on the reaction equilibrium is denoted as an equilibrium isotope effect (EIE). In particular, KIE is an extremely useful tool in chemistry and biology, and has been used extensively in enzymology [12][13][14][15][16]. In the case of reactions with a single transition state (TS) separating reactants and products, the experimentally observed KIEs provide direct information regarding the change in bonding during the chemical event.…”

Section: Introductionmentioning

confidence: 99%