Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes

Herschlag, Daniel; Natarajan, Aditya

doi:10.1021/bi4000113

Cited by 80 publications

(108 citation statements)

References 171 publications

(308 reference statements)

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“…Ketosteroid isomerase (KSI) is a small, proficient enzyme with one of the highest known unimolecular rate constants in biochemistry (1, 2), which has prompted extensive study of its mechanism and the catalytic strategies it uses (3–5). In steroid biosynthesis and degradation, KSI alters the position of a C=C double bond (Fig.…”

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confidence: 99%

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Fried

Bagchi

Boxer

2014

Science

444

641

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Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

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confidence: 99%

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Fried

Bagchi

Boxer

2014

Science

444

641

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“…Ketosteroid isomerase (KSI) possesses one of the highest enzyme unimolecular rate constants and thus, is considered a paradigm of proton transfer catalysis in enzymology (5)(6)(7)(8)(9)(10)(11). The remarkable rate of KSI is intimately connected to the formation of a hydrogen-bond network in its active site (Fig.…”

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confidence: 99%

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

Wang

Fried

Boxer

et al. 2014

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

132

178

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Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.enzyme | hydrogen bonding | nuclear quantum effects | proton delocalization | ab initio path integral molecular dynamics A lthough many biological processes can be well-described with classical mechanics, there has been much interest and debate as to the role of quantum effects in biological systems ranging from photosynthetic energy transfer, to photoinduced isomerization in the vision cycle and avian magnetoreception (1). For example, nuclear quantum effects, such as tunneling and zero-point energy (ZPE), have been observed to lead to kinetic isotope effects of greater than 100 in biological proton and proton-coupled electron transfer processes (2, 3). However, the role of nuclear quantum effects in determining the groundstate thermodynamic properties of biological systems, which manifest as equilibrium isotope effects, has gained significantly less attention (4).Ketosteroid isomerase (KSI) possesses one of the highest enzyme unimolecular rate constants and thus, is considered a paradigm of proton transfer catalysis in enzymology (5-11). The remarkable rate of KSI is intimately connected to the formation of a hydrogen-bond network in its active site (Fig. 1A), which acts to stabilize a charged dienolate intermediate, lowering its free energy by ∼11 kcal/mol (1 kcal = 4.18 kJ) relative to solution (Fig. S1) (6). This extended hydrogen-bond network in the active site links the substrate to Asp103 and Tyr16, with the latter further hydrogen-bonded to Tyr57 and Tyr32, which is shown in Fig. 1A.The mutant KSI D40N preserves the structure of the wild-type (WT) enzyme while mimicking the protonation state of residue 40 in the intermediate complex (Fig. 1B), therefore permitting experimental investigation of an intermediate-like state of the enzyme (6,(12)(13)(14). Experiments have identified that, in the absence of an inhibitor, one of the residues in the active site of KSI D40N is deprotonated (12). Although one might expect the carboxylic acid of Asp103 to be deprotonated, the combination of recent 13 C NMR and ultraviolet visible spectroscopy (UVVis) e...

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“…Nevertheless, due to the complexity of even the simplest enzymatic systems, a quantitative understanding of the energetics of catalysis has remained elusive. The relative contributions of electrostatic, steric, proximity, and solvent effects to transition state stabilization and reactant destabilization, as well as the role of dynamic motions and tunneling, are still heatedly debated (6)(7)(8)(9)(10).…”

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confidence: 99%

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

Burschowsky

Eerde

Ökvist

et al. 2014

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

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For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.pericyclic reaction | catalysis | enzyme mechanism | near attack conformation | X-ray crystal structures

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Fundamental Challenges in Mechanistic Enzymology: Progress toward Understanding the Rate Enhancements of Enzymes

Cited by 80 publications

References 171 publications

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis

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