Engineering a model protein cavity to catalyze the Kemp elimination

Merski, M.; Shoichet, Brian K.

doi:10.1073/pnas.1208076109

Cited by 50 publications

(54 citation statements)

References 41 publications

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“…It appears that the inherent conformational flexibility of proteins, water penetration into the core, the unavoidable presence of backbone polar atoms, and favorable Coulomb interactions are sufficient to stabilize a charge cluster buried in the relatively dry protein interior. This study provides a clear demonstration that one of the most important factors that has to be considered in rational design or evolution of novel enzymatic active sites is the need for a high thermodynamic stability sufficient to allow the protein to withstand the unavoidable, destabilizing consequences associated with the presence of ionizable residues in hydrophobic environments (41).…”

Section: Discussionmentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

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

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An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK a values near 7. When buried together their pK a values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.electrostatics | internal charge | low-barrier hydrogen bond | dielectric constant | enzyme design P aired ionizable groups buried in hydrophobic environments inside proteins are an uncommon but essential structural motif necessary for H + transport (1), e − transfer (2), catalysis (3-5), and other important biochemical processes. Despite their importance, the properties of these buried ionizable pairs are poorly understood because they are difficult to study experimentally in the proteins where they play functional roles. When the pair consists of a basic and an acidic group it is usually assumed to be charged, but this is often just speculation without direct experimental support (6, 7). In principle, a Coulomb interaction between two ionizable groups of opposite polarity buried in a dehydrated environment inside a protein can be very strong, but this has not been established experimentally. Simulations suggest that buried ion pairs are always destabilizing (8); however, from an experimental perspective it is not known if the favorable Coulomb interaction would be sufficient to compensate for the unfavorable dehydration of the two buried charges or if the net effect of the pair on the thermodynamic stability of a protein would be stabilizing or not. These issues were examined in detail with an artificial ion pair buried in the hydrophobic interior of staphylococcal nuclease (SNase).The buried ion pair was engineered by introducing the V23E and L36K substitutions in a highly stable form of SNase. The pK a values of Glu-23 and Lys-36 introduced individually are anomalous (pK a of 7.1 for ref. 9; ref. 10), consistent with the groups existing in hydrophobic environments that are neither as polar nor as polarizable as water (11-13). Structural modeli...

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Section: Discussionmentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

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

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“…Using simple modeling Merski and Shoichet introduced a histidine residue into an engineered mutant of a T4 lysozyme to create a Kemp eliminase [20]. Subsequent rational improvement of the catalyst guided by structural information yielded an enzyme with a catalytic efficiency of 1.8 m −1 s −1 at pH 5.0, and the structure of the protein was confirmed by X-ray crystallography.…”

Section: The Kemp Eliminationmentioning

confidence: 99%

Catalytic efficiency of designed catalytic proteins

Korendovych

DeGrado

2014

Current Opinion in Structural Biology

108

106

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The de novo design of catalysts that mimic the affinity and specificity of natural enzymes remains one of the Holy Grails of chemistry. Despite decades of concerted effort we are still unable to design catalysts as efficient as enzymes. Here we critically evaluate approaches to (re)design of novel catalytic function in proteins using two test cases: Kemp elimination and ester hydrolysis. We show that the degree of success thus far has been modest when the rate enhancements seen for the designed proteins are compared with the rate enhancements by small molecule catalysts in solvents with properties similar to the active site. Nevertheless, there are reasons for optimism: the design methods are ever improving and the resulting catalyst can be efficiently improved using directed evolution.

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“…For more than two decades2, Kemp eliminases based on various protein scaffolds have provided valuable insights into understanding and mimicking enzymes23456789101112131415 (Fig. 1a).…”

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confidence: 99%

A redox-mediated Kemp eliminase

Wang

Ilie

et al. 2017

Nat Commun

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The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N–O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)–N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.

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Engineering a model protein cavity to catalyze the Kemp elimination

Cited by 50 publications

References 41 publications

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Catalytic efficiency of designed catalytic proteins

A redox-mediated Kemp eliminase

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