Steady-state analysis of kinetic isotope effects in enzymic reactions

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, 13 C−, 15 N−, 2 H−labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing. femtosecond motions | kinetic isotope effect | transition state structure | enzymatic catalysis | protein dynamics

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“…E-mail: vern.schramm@einstein.yu.edu. where L V ∕K is the observed isotope effect on V ∕K and L k is the intrinsic isotope effect (23).…”

Section: Resultsmentioning

confidence: 99%

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

Silva

Murkin

Schramm

2011

Proc. Natl. Acad. Sci. U.S.A.

114

204

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“…(v) Intrinsic kinetic isotope effects The intrinsic D/T KIEs were derived by numerically solving the following equation (Northrop 1975(Northrop , 1977(Northrop , 1991Kohen 2003Kohen , 2005Cleland 2005 (Streitwieser et al 1958;Kohen 2003). In the procedure, it is assumed that the Swain-Schaad relationship holds or has little temperature dependence for intrinsic primary KIEs (Northrop 1991;Francisco et al 2002;Kohen 2003;Cleland 2005).…”

Section: Methodsmentioning

confidence: 99%

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Wang

Goodey

Benkovic

et al. 2006

Phil. Trans. R. Soc. B

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Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor-acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface prearrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.

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“…Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3,4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5)(6)(7)(8)(9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10)(11)(12).…”

mentioning

confidence: 99%

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

Linscott

Kapilashrami

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

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Steady-state analysis of kinetic isotope effects in enzymic reactions

Cited by 395 publications

References 13 publications

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

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