Kinetic .alpha.-deuterium isotope effects for acylation of chymotrypsin by 4-methoxyphenyl formate and for deacylation of formylchymotrypsin

Lehrmann, G.; Quinn, Dan; Cordes, E. H.

doi:10.1021/ja00527a075

Cited by 6 publications

(6 citation statements)

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“…The magnitude of the KIE for fluoroacetyl-CoA hydrolysis is on the order of or larger than KIEs measured for other characterized enzymes that catalyze C α deprotonation (34,35). In comparison, enzymes that carry out nucleophilic attack at a carbonyl moiety typically show an inverse isotope effect, if any, with similarly labeled substrates (36). To further confirm the ability of FlK to initiate C α deprotonation, we assayed the 3,3,3-trifluoropropionyl-CoA (14) observed rapid formation of one equivalent of CoA followed by elimination of one equivalent of fluoride, suggesting formation of an enolate or carbanion species (37) (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 83%

Catalytic control of enzymatic fluorine specificity

Weeks¹,

Chang

2012

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

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The investigation of unique chemical phenotypes has led to the discovery of enzymes with interesting behaviors that allow us to explore unusual function. The organofluorine-producing microbe Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that demonstrates a surprisingly high level of discrimination for a single fluorine substituent on its substrate compared with the cellularly abundant hydrogen analog, acetyl-CoA. In this report, we show that the high selectivity of FlK is achieved through catalysis rather than molecular recognition, where deprotonation at the C α position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10 4 -fold compared with the nonfluorinated congener. These studies provide insight into mechanisms of catalytic selectivity in a native system where the existence of two reaction pathways determines substrate rather than product selection.enzyme mechanism | substrate selectivity L iving organisms have solved some of the most difficult challenges in catalysis by harnessing the exquisite selectivity and reactivity of enzyme active sites to build complex chemical behaviors (1-5). Indeed, the plasticity of enzymes toward evolution of new function has allowed life to flourish in diverse biospheres by conferring a selective advantage to hosts that can demonstrate catalytic innovation and use unusual resources. The enzymes that form the molecular basis for these chemical phenotypes are a rich source of molecular diversity and provide an experimental platform for the continual search to gain insight into the mechanisms and dynamics of protein and organismal evolution (6-9). One of the salient features of enzymes is their extremely high substrate selectivity, which allows them to choose the correct substrate over the thousands of other small molecules that are present in cells at any given time. Thus, the exploration of substrate specificity and its evolution is key for understanding both enzyme catalysis and the expansion of biodiversity at the molecular level (7)(8)(9)(10)(11)(12).In this context, a singular chemical trait found in the soil bacterium Streptomyces cattleya is its ability to catalyze the formation of carbon-fluorine bonds (13,14). Fluorine resides at one corner of the periodic table, and its unique elemental properties make it highly effective as a design element for drug discovery but also quite challenging for the synthesis of fluorinecontaining molecules (15)(16)(17). Although fluorine has emerged as a common motif in synthetic compounds, including top-selling drugs such as atorvastatin and fluticasone, it has been underexploited in nature, and only a few biogenic organofluorine compounds (<20) have been identified to date despite the thousands of known natural products containing chlorine and bromine (∼5,000) (18,19). In fact, the only fully characterized organofluorine natural products are derived from a single pathway that produces the deceptively simple poison fluoroacetate (1). T...

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Section: Resultsmentioning

confidence: 83%

Catalytic control of enzymatic fluorine specificity

Weeks¹,

Chang

2012

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

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“…It is expected to fall in the range from unity to EIE. When it is closer to unity, the TS is reactant-like; when it is closer to EIE, the TS is product-like. ,− This empirical prediction, however, often fails for H-transfer reactions, especially in enzymes. One of the earliest observations was by Cleland and co-workers who found a normal α-2° KIE of 1.23 at the 4-H/D position of NAD + in its oxidation of the formate ion via hydride transfer catalyzed by a formate dehydrogenase .…”

Section: Introductionmentioning

confidence: 99%

Imbalanced Transition States from α-H/D and Remote β-Type N-CH/D Secondary Kinetic Isotope Effects on the NADH/NAD⁺ Analogues in Their Hydride Tunneling Reactions in Solution

Sakhaee

Jafari

et al. 2019

J. Org. Chem.

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The α-H/D (if available) and remote β-type N-CH 3 /CD 3 2°kinetic isotope effects (KIEs) on 10-methylacridine (MAH), 9,10-dimethylacridine (DMAH), 1,3-dimethyl-2-phenylbenzimidazoline (DMPBIH) and on the oxidized forms MA + and DMA + , in their hydride transfer reactions with several hydride acceptors/donors in acetonitrile, were determined. The corresponding equilibrium isotope effects (EIEs) were computed. Hammett correlations of several closely related hydride transfer reactions were constructed using the literature data. The α-2°KIEs on both MAH and MA + are inflated relative to the semiclassical prediction on the basis of the KIE/EIE comparison and Hammond's postulate. This together with previously published unusual 1°and 2°K IE behaviors strongly suggest a H-tunneling mechanism. By comparing with the EIEs, the α-2°KIEs were used to analyze the rehybridization of the reaction center C and the N-CH 3 /CD 3 2°KIEs to calculate the charge distribution on the structure containing N during H-tunneling. The rehybridization appears to lag behind the charge development in the donor moiety. The charge distribution at the tunneling ready transition state is in agreement with the Hammett correlations; the donor is productlike, and the acceptor is reactant-like, indicative of a partial negative charge borne by the "in-flight" nucleus being "hydridic" in character. Results were compared with the α-2°KIEs on NADH/NAD + and the Hammett correlations in closely related enzymes. The comparison implicates that the H-tunneling probability would be enhanced by these enzymes.

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“…A particularly interesting quantity is the magnitude of a 2° KIE relative to the corresponding equilibrium isotope effect (2° EIE), which is the ratio of the isotopically unsubstituted equilibrium constant to the isotopically substituted one. − For an atom transfer reaction, where the 2° isotopic atom is bonded to the donor (or the acceptor atom), the 2° KIEs are often considered to be mainly a consequence of the change in the hybridization state of donor or acceptor in proceeding from the reactant to the transition state. , Such hybridization changes have important effects on bending and stretching frequencies between the reactant and the transition state. ,− On the basis of this relationship between hybridization and KIEs, an empirical criterion has been widely used to infer the location of the transition state by comparing the 2° KIE for deuterium substitution to the 2° EIE. Thus a 2° KIE that is close to the 2° EIE is taken as an indication that the rehybridization of the reaction center bonded to the deuterium has already been accomplished at the transition state, yielding a late transition state that resembles the product. ,,− ,− The same criterion was also used to suggest that a small 2° KIE (close to unity) results from an early transition state (i.e., one resembling the reactant), and the fractional position of a 2° KIE between unity and the relevant 2° EIE ([2° KIE − 1]/[2° EIE − 1]) represents the fractional location of the transition state between reactant and product. ,,− However, this criterion seems to be oversimplified by neglecting factors other than the structural change from the reactant to the transition state that can significantly contribute to 2° KIEs. ,, Simply comparing the magnitude of the 2° KIE to that of the 2° EIE without considering such factors may lead to an incorrect characterization of the transition state structure.…”

Section: Introductionmentioning

confidence: 99%

“…Thus a 2°KIE that is close to the 2°EIE is taken as an indication that the rehybridization of the reaction center bonded to the deuterium has already been accomplished at the transition state, yielding a late transition state that resembles the product. 1,9,[13][14][15][16][22][23][24][25][26][27][28][29] The same criterion was also used to suggest that a small 2°KIE (close to unity) results from an early transition state (i.e., one resembling the reactant), and the fractional position of a 2°KIE between unity and the relevant 2°EIE ([2°KIE -1]/[2°EIE -1]) represents the fractional location of the transition state between reactant and product. 9,16,[22][23][24][25][26][27][28][29][30] However, this criterion seems to be oversimplified by neglecting factors other than the structural change from the reactant to the transition state that can significantly contribute to 2°KIEs.…”

Section: Introductionmentioning

confidence: 99%

“…1,9,[13][14][15][16][22][23][24][25][26][27][28][29] The same criterion was also used to suggest that a small 2°KIE (close to unity) results from an early transition state (i.e., one resembling the reactant), and the fractional position of a 2°KIE between unity and the relevant 2°EIE ([2°KIE -1]/[2°EIE -1]) represents the fractional location of the transition state between reactant and product. 9,16,[22][23][24][25][26][27][28][29][30] However, this criterion seems to be oversimplified by neglecting factors other than the structural change from the reactant to the transition state that can significantly contribute to 2°KIEs. 18,19,31 Simply comparing the magnitude of the 2°K IE to that of the 2°EIE without considering such factors may lead to an incorrect characterization of the transition state structure.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Nonperfect Synchronization of Reaction Center Rehybridization in the Transition State of the Hydride Transfer Catalyzed by Dihydrofolate Reductase

Garcia‐Viloca

et al. 2005

J. Am. Chem. Soc.

109

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It has been suggested that the magnitudes of secondary kinetic isotope effects (2 degrees KIEs) of enzyme-catalyzed reactions are an indicator of the extent of reaction-center rehybridization at the transition state. A 2 degrees KIE value close to the corresponding secondary equilibrium isotope effects (2 degrees EIE) is conventionally interpreted as indicating a late transition state that resembles the final product. The reliability of using this criterion to infer the structure of the transition state is examined by carrying out a theoretical investigation of the hybridization states of the hydride donor and acceptor in the Escherichia coli dihydrofolate reductase (ecDHFR)-catalyzed reaction for which a 2 degrees KIE close to the 2 degrees EIE was reported. Our results show that the donor carbon at the hydride transfer transition state resembles the reactant state more than the product state, whereas the acceptor carbon is more productlike, which is a symptom of transition state imbalance. The conclusion that the isotopically substituted carbon is reactant-like disagrees with the conclusion that would have been derived from the criterion of 2 degrees KIEs and 2 degrees EIEs, but the breakdown of the correlation with the equilibrium isotope effect can be explained by considering the effect of tunneling.

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Kinetic .alpha.-deuterium isotope effects for acylation of chymotrypsin by 4-methoxyphenyl formate and for deacylation of formylchymotrypsin

Cited by 6 publications

References 4 publications

Catalytic control of enzymatic fluorine specificity

Catalytic control of enzymatic fluorine specificity

Imbalanced Transition States from α-H/D and Remote β-Type N-CH/D Secondary Kinetic Isotope Effects on the NADH/NAD⁺ Analogues in Their Hydride Tunneling Reactions in Solution

Nonperfect Synchronization of Reaction Center Rehybridization in the Transition State of the Hydride Transfer Catalyzed by Dihydrofolate Reductase

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Kinetic .alpha.-deuterium isotope effects for acylation of chymotrypsin by 4-methoxyphenyl formate and for deacylation of formylchymotrypsin

Cited by 6 publications

References 4 publications

Catalytic control of enzymatic fluorine specificity

Catalytic control of enzymatic fluorine specificity

Imbalanced Transition States from α-H/D and Remote β-Type N-CH/D Secondary Kinetic Isotope Effects on the NADH/NAD+ Analogues in Their Hydride Tunneling Reactions in Solution

Nonperfect Synchronization of Reaction Center Rehybridization in the Transition State of the Hydride Transfer Catalyzed by Dihydrofolate Reductase

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Imbalanced Transition States from α-H/D and Remote β-Type N-CH/D Secondary Kinetic Isotope Effects on the NADH/NAD⁺ Analogues in Their Hydride Tunneling Reactions in Solution