Experimental Evidence for Enzyme-Enhanced Coupled Motion/Quantum Mechanical Hydrogen Tunneling by Ketosteroid Isomerase

Wilde, Thomas; Błotny, Grzegorz; Pollack, Ralph M.

doi:10.1021/ja0732330

Cited by 22 publications

(43 citation statements)

References 56 publications

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“…Indeed, primary and secondary isotope effects for KSI, our highlighted enzyme above, are different in the corresponding solution (acetate-catalyzed) and enzyme-catalyzed reactions and consistent with enhanced coupled motion of the substrate along the reaction coordinate in the enzymatic reaction (156). But it is difficult to ascertain whether the changes in reaction properties in this and other enzymatic reactions are causative for catalysis.…”

Section: Outlook and Future Questionsmentioning

confidence: 67%

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

2013

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Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multi-faceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.

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Section: Outlook and Future Questionsmentioning

confidence: 67%

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

2013

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“…These results also provide no indication of coupling of proton transfer, which involves Asp40 as a general base, to the electrostatic properties of the oxyanion hole hydrogen bond donor. 65 …”

Section: Resultsmentioning

confidence: 99%

Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site

2014

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Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr’s) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.

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“…The term tunneling and coupled motion has been applied to ADHs and other systems that exhibit 2°KIEs outside the semiclassical range (12,28,37,38), as well as reactions that violate the Rule of the Geometric Mean, as evidenced by inflated mSSEs (1,18,21). The term tunneling and coupled motion appears to be used for both abnormalities in 2°KIEs.…”

Section: Resultsmentioning

confidence: 99%

Elusive transition state of alcohol dehydrogenase unveiled

Roston

Kohen

2010

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

154

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For several decades the hydride transfer catalyzed by alcohol dehydrogenase has been difficult to understand. Here we add to the large corpus of anomalous and paradoxical data collected for this reaction by measuring a normal (>1) 2°kinetic isotope effect (KIE) for the reduction of benzaldehyde. Because the relevant equilibrium effect is inverse (<1), this KIE eludes the traditional interpretation of 2°KIEs. It does, however, enable the development of a comprehensive model for the "tunneling ready state" (TRS) of the reaction that fits into the general scheme of Marcus-like models of hydrogen tunneling. The TRS is the ensemble of states along the intricate reorganization coordinate, where H tunneling between the donor and acceptor occurs (the crossing point in Marcus theory). It is comparable to the effective transition state implied by ensemble-averaged variational transition state theory. Properties of the TRS are approximated as an average of the individual properties of the donor and acceptor states. The model is consistent with experimental findings that previously appeared contradictory; specifically, it resolves the long-standing ambiguity regarding the location of the TRS (aldehyde-like vs. alcohol-like). The new picture of the TRS for this reaction identifies the principal components of the collective reaction coordinate and the average structure of the saddle point along that coordinate.hydrogen tunneling | enzyme kinetic | secondary isotope effect | Swain-Schaad E nzymes enhance the rates of chemical reactions by many orders of magnitude, and extensive studies have uncovered many aspects of how they do so. The prevailing theory that enzymes stabilize the transition state (TS) relative to the reactants explains many phenomena, and a great deal of contemporary research focuses on how enzymes stabilize the TS. An unfortunate hindrance to developing an understanding of how enzymes stabilize the TS lies in the enigmatic nature of the TS. Many enzymatic hydrogen (proton, hydrogen, or hydride) transfers, for example, occur by quantum mechanical tunneling, where a particle passes through an energy barrier because of its wave properties (1-6).Yeast alcohol dehydrogenase (yADH) serves as an excellent model system for enzyme-catalyzed H transfer because unlike many other enzymes, the chemical step, oxidation of a primary alcohol to an aldehyde by NAD þ , is rate-limiting with aromatic substrates (7). Furthermore, the thermodynamics of the reaction allow for examination of both the forward (alcohol to aldehyde) and reverse (aldehyde to alcohol) reactions under similar conditions (8). This type of nicotinamide-dependent redox reaction appears ubiquitously in biology, and a detailed picture of the TS of such reactions may facilitate many medical and industrial applications.Intriguingly, decades of experiments on the reaction catalyzed by yADH have returned what appear to be contradictory results, some suggesting an early TS, whereas others point to a late TS (7-10). In pioneering studies, Klinman measured linear fr...

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Experimental Evidence for Enzyme-Enhanced Coupled Motion/Quantum Mechanical Hydrogen Tunneling by Ketosteroid Isomerase

Cited by 22 publications

References 56 publications

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

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

Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site

Elusive transition state of alcohol dehydrogenase unveiled

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