The Expression of Isotope Effects on Enzyme-Catalyzed Reactions

Northrop, Dexter B.

doi:10.1146/annurev.bi.50.070181.000535

Cited by 336 publications

(368 citation statements)

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“…Even though substrate binding and product release is unchanged in heavy PNP, the probability of chemical barrier crossing for the Michaelis complexes is decreased in both forward commitment and single-turnover experiments. Forward commitment, C f , is the probability of a substrate in the Michaelis complex to pass the chemical barrier relative to its probability of dissociation to free substrate (31). The lowered C f for inosine and guanosine with heavy PNP reflects a decrease in the probability of on-enzyme barrier crossing.…”

Section: Discussionmentioning

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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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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Section: Discussionmentioning

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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“…12-14, c fb and c fc (forward commitments to catalysis for B and C) are , and , respectively, c r (reverse commitment to catalysis) is , and c vf (the catalytic ratio) is (16).…”

Section: Primary Substrate Deuterium Kinetic Isotope Effectsmentioning

confidence: 99%

A Proposed Proton Shuttle Mechanism for Saccharopine Dehydrogenase from Saccharomyces cerevisiae

Alguindigue

West

et al. 2006

Biochemistry

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Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming)) catalyzes the final step in the α-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate α-Kg and lysine using NAD + as an oxidizing agent. Proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD and saccharopine bind, a group with a pK a of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pK a of 7.2 accepts a proton from H 2 O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate α-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V 2 /K NADH , consistent with a steady state random mechanism, and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect of D V 2 and D (V 2 /K Lys ), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effect, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V 2 and V 2 /K Lys suggesting proton transfer exists in at least two sequential transition states.Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming); (EC 1.5.1.7)) (SDH 1 ) catalyzes the final step in the α-aminoadipate pathway (AAA) for the de novo synthesis of L-lysine in fungi (1,2). The enzyme catalyzes the reversible † This work is supported by the Grayce B. Kerr Endowment to the University of Oklahoma (to P.F.C.), and a grant (GM 071417) from the National Institute of Health (to P.F.C. and A.H.W.). * Corresponding author: E-mail: pcook@chemdept.chem.ou.edu 1 Abbreviations: SDH, saccharopine dehydrogenase; AAA, α-aminoadipate pathway; α-Kg, α-ketoglutarate; Sacc,...

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“…In our particular case, where the reaction scheme can also be entered via cADPR (step k −& ), if k −% k & one expects that on hydrolysis of the cyclic metabolite the CD38-cADPR complex, which now corresponds to a Michaelis complex, has a high commitment to catalysis. Forward commitment (C f ) to catalysis has been defined as the tendency of the enzyme-substrate complex poised for catalysis to continue forward as opposed to its tendency to partition back into free enzyme and substrate [25]. In the case of a high commitment to catalysis k cat k off ; in Scheme 1 this translates into k −% k & for the catalytic pathway leading to hydrolysis of cADPR, because, in the reactions catalysed by bovine spleen NAD + glycohydrolase, the hydrolytic step (k $ ) is fast compared with the bond cleavage steps [26,27].…”

Section: Commitment To Catalysis Of the Cd38-cadpr Complexmentioning

confidence: 99%

Kinetic competence of the cADP-ribose‒CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling

et al. 2001

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The Expression of Isotope Effects on Enzyme-Catalyzed Reactions

Cited by 336 publications

References 1 publication

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

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

A Proposed Proton Shuttle Mechanism for Saccharopine Dehydrogenase from Saccharomyces cerevisiae

Kinetic competence of the cADP-ribose‒CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling

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