Characterization of Proton-Transfer Catalysis by Serum Albumins

Hollfelder, Florian; Kirby, Anthony J.; Tawfik, Dan S.; Kikuchi, and Kazuya; Hilvert, Donald

doi:10.1021/ja993471y

Cited by 75 publications

(98 citation statements)

References 70 publications

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“…The reaction, shown in Fig. 1a, has been extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon-a process that is normally restricted by high activation-energy barriers 7,8 .…”

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confidence: 99%

Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

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The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10 5 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a .200-fold increase in k cat /K m (k cat /K m of 2,600 M 21 s 21 and k cat /k uncat of .10 6 ). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.Naturally occurring enzymes are extraordinarily efficient catalysts 1 . They bind their substrates in a well-defined active site with precisely aligned catalytic residues to form highly active and selective catalysts for a wide range of chemical reactions under mild conditions. Nevertheless, many important synthetic reactions lack a naturally occurring enzymatic counterpart. Hence, the design of stable enzymes with new catalytic activities is of great practical interest, with potential applications in biotechnology, biomedicine and industrial processes. Furthermore, the computational design of new enzymes provides a stringent test of our understanding of how naturally occurring enzymes work. In the past several years, there has been exciting progress in designing new biocatalysts 2,3 .Here we describe the use of our recently developed computational enzyme design methodology 4 to create new enzyme catalysts for a reaction for which no naturally occurring enzyme exists: the Kemp elimination 5,6 . The reaction, shown in Fig. 1a, has been extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon-a process that is normally restricted by high activation-energy barriers 7,8 . Computational design methodThe first step in our protocol for designing new enzymes is to choose a catalytic mechanism and then to use quantum mechanical transition state calculations to create an idealized active site with protein functional groups positioned so as to maximize transition state stabilization (Fig. 1b). The key step for the Kemp elimination is deprotonation of a carbon by a general base. We chose two different catalytic bases for this purpose: first, the carboxyl group of an aspartate or glutamate side chain, and, second, the imidazole of a histidine positioned and polarized by the carboxyl group of an aspartate or glutamate (we refer to this combination as a His-Asp dyad). The two choices have complementary strengths and weaknesses. The advantage of the carboxylate...

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confidence: 99%

Kemp elimination catalysts by computational enzyme design

Röthlisberger¹,

Khersonsky²,

Wollacott³

et al. 2008

Nature

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1,187

1,237

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“…computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2)(3)(4)(5)(6)(7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig.…”

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confidence: 99%

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Khersonsky

Kiss

Röthlisberger

et al. 2012

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

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Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cat ∕K m values of ≤5 × 10 4 M −1 s −1 ). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cat ∕K m , and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher k cat values. The best KE59 variants exhibited k cat ∕K m values up to 0.6 × 10 6 M −1 s −1 , and k cat ∕k uncat values of ≤10 7 almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2-7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig. 1A). Activation of a carboxylate base catalyst by desolvation is efficiently mimicked by aprotic dipolar solvents such as acetonitrile and by various enzyme mimics. Effective alignment of the substrate and charge-dispersing interactions that stabilize the negatively charged transition state (TS) have also been achieved by various enzyme mimics that catalyze the Kemp elimination (8, 9). However, generation of a (wo)manmade active site that exhibits all these features and performs as well as natural enzymes, remains a challenge.Computational methods for predicting structure from sequence at atomic accuracy provide a new approach to enzyme engineering (10-12). The design involves two steps: (i) designing an active-site configuration that may confer efficient catalysis, (ii) computing a sequence that confers the desired configuration. Both steps are currently far from optimal, as the catalytic efficiency of designed enzymes falls far behind that of natural enzymes. Further, as with other enzyme mimics, computational designs tackle activated model systems and fail to catalyze cha...

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“…These criteria were fulfilled by calmodulin (CaM), a prototypical member of the EF-hand class of regulatory proteins, and we chose its C-terminal domain (cCaM, M76-K148) for its stability and high affinity for Ca 2þ (17,18). Importantly, CaM is known to have no catalytic activity in the Kemp elimination reaction (19).…”

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Design of a switchable eliminase

Korendovych

Kulp

et al. 2011

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

118

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The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.protein design | catalysis | pKa T he design of enzyme-like catalytic proteins is a challenging endeavour that has been approached using combinatorial (1) and computational strategies (2), as well as by following simple chemical principles (3, 4). The Kemp elimination (5) (Scheme 1) is an extensively studied benchmark for catalyst design (6-8) as a model for enzymatic C-H bond abstraction. Previously, computational methods were employed to alter the active sites of natural enzymes to catalyze this reaction (7). Mutation of a dozen or more side chains resulted in catalysts that showed rate enhancements of 10 4 to 10 5 relative to a given reference rate. Here, by combining a few physical principles with modern computational tools, we show that a single mutation can confer similar catalytic efficiency into a much simpler and allosterically switchable scaffold that previously had no catalytic activity. This approach circumvents often contentious questions concerning residual activities of the enzymatic framework or the appropriate reference background rate for expression of a "rate enhancement" while also suggesting a practical approach to the design of catalytically amplified sensors.Here we applied a minimalist approach to protein design (9-11), which emphasizes the use of the simple scaffolds and the min...

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Characterization of Proton-Transfer Catalysis by Serum Albumins

Cited by 75 publications

References 70 publications

Kemp elimination catalysts by computational enzyme design

Kemp elimination catalysts by computational enzyme design

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Design of a switchable eliminase

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