Enzymatic transition states and dynamic motion in barrier crossing

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201

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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confidence: 86%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Silva

Murkin

Schramm

2011

114

201

“…Slower (millisecond) motions are involved in conformational changes that occur during substrate binding and product release (1)(2)(3)(4), whereas femtosecond motions (promoting vibrations) are proposed by computational studies to be involved in the chemical step of transition-state formation (covalent bond changes) (5)(6)(7)(8). The role of fast (femtosecond) enzyme dynamics in the catalytic cycle has remained elusive and controversial due to the experimental challenge of probing femtosecond motions in enzymes.…”

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confidence: 99%

Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

Suárez

Schramm

2015

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP ( 13 C, 15 N, and 2 H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both heavy enzymes | transition state coupling | femtosecond dynamics | pre-steady-state chemistry | Born-Oppenheimer enzymes P rotein and atomic motions required for enzymatic catalysis involve a broad range of timescales from the millisecond to the femtosecond. Slower (millisecond) motions are involved in conformational changes that occur during substrate binding and product release (1-4), whereas femtosecond motions (promoting vibrations) are proposed by computational studies to be involved in the chemical step of transition-state formation (covalent bond changes) (5-8). The role of fast (femtosecond) enzyme dynamics in the catalytic cycle has remained elusive and controversial due to the experimental challenge of probing femtosecond motions in enzymes. Previous studies from our laboratory and others have reported that isotopic substitution to create heavy enzymes ( 13 C, 15 N, and 2 H) often slows catalytic site chemistry and, in some cases, alters rate constants for nonchemical steps (9-12). Reduced catalytic site barrier-crossing (the chemical step) in isotopically labeled enzymes supports coupling of local femtosecond motion to transition-state formation and, in some cases, interaction with slower modes (10, 11).In early heavy-enzyme studies, Silva et al. (9) uniformly labeled the amino acid residues of human purine nucleoside phosphorylase (PNP) with 13 C, 15 N, and nonexchangeable 2 H in an attempt to perturb local bond vibrational modes without altering the electrostatics of the enzyme (the Born-Oppenheimer approximation) (13). Heavy PNP demonstrated unchanged steady-state kinetic parameters, kinetic isotope effects, and transition state structure, but the chemical rate was decreased. Transition path sampling with heavy and light PNPs studied reactive trajectories and predicted that femtosecond vibrational motions are less coupled in the heavy enzyme (14).PNP (EC 2.4.2.1) catalyzes the reversible phosphorolysis of 6-oxypurine (and 2′-deoxy)-β-D-ribonucleosides to yield (2-deoxy)-α-D-ribose 1-phosphate and the purine base (15) (Fig. 1). Isotopically labeled PNP expressed from 13 C and 15 N precursors in D 2 O solvent is 9.9% heavier than natural abundance PNP and shows a heavy enzyme kinetic isotope effect (KIE) of 1.36 (k chem light/k chem heavy) (9). Reduced catalytic site ch...

“…The origins of the enormous catalytic power of enzymes are still not well understood despite enormous effort (1)(2)(3)(4)(5)(6). In particular, the functional role of fast picosecond protein motions in catalysis and the dynamic nature of the transition state barrier crossing is a subject of ongoing and current debate (7)(8)(9)(10)(11)(12)(13). Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22).…”

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confidence: 99%

Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Jha¹,

Gaffney

et al. 2011

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ 5 -3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.enzyme catalysis | electrostatic preorganization | Stark effect | visible-pump IR probe | time-resolved anisotropy E nzymes catalyze the vast majority of biochemical reactions and accelerate the rate of these reactions by many orders of magnitude compared to the uncatalyzed reactions in solution. The origins of the enormous catalytic power of enzymes are still not well understood despite enormous effort (1-6). In particular, the functional role of fast picosecond protein motions in catalysis and the dynamic nature of the transition state barrier crossing is a subject of ongoing and current debate (7-13). Theoretical studies have suggested that fast vibrations in enzymes might generate transition state conformations conducive to the chemical reaction (7,8,(14)(15)(16)(17)(18)(19)(20)(21), a familiar concept for reactions in ordinary solvents (22). In an alternative viewpoint, preorganization effects have been suggested to be a major contributing factor to enzyme catalysis (23-28). It has been postulated that enzymes have partially oriented dipoles of polar and charged groups in the active site that interact with electrostatic features present in the catalytic transition state more favorably than water can. Such preorganization of active site dipoles and charges has been suggested to result in a reduction in the reorganization energy and provide enormous catalytic advantage over the reaction in solution, where water molecules must rearrange in order to solvate charge rearrangements during the chemical reaction (24,(29)(30)(31). The relative merits of these opposing viewpoints remains unclear largely because of the paucity of direct experimental assessment of the key discrepancies betwee...