Cooperative Nucleophilic and General-Acid Catalysis by the HisH<sup>+</sup>−His Pair and Arginine Transition State Binding in Catalysis of Ester Hydrolysis Reactions by Designed Helix−Loop−Helix Motifs

Broo, Klas; Nilsson, Helena; Nilsson, Jonas; Flodberg, and Anna; Baltzer, Lars

doi:10.1021/ja9737580

Cited by 72 publications

(65 citation statements)

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“…[29] In work involving designed helix-loop-helix catalysts, it has been shown that a His-His pair in an (i, i+4) configuration can accomplish the cooperative catalysis of para-nitrophenyl ester hydrolysis. [9] The creation of the F131H/V135H variant represents a grafting of the original His-His motif from the helix-loop-helix construct into HCAII, by placing histidines in a helical portion of the enzyme in relative positions so that an (i, i+4) configuration is preserved.…”

Section: Grafting Of a Helix Two-histidine Catalytic Site Into Hcaiimentioning

confidence: 99%

“…In their work on de novo designed helix-loop-helix polypeptides, Baltzer and his group have constructed a catalytic site for ester hydrolysis consisting of two histidines, acting cooperatively. [9] Bolon and Mayo used a computational approach to choose mutations for binding and histidine-mediated nucleophilic hydrolysis of an activated ester by E. coli thioredoxin, resulting in a mutant with kinetic parameters comparable to those of the early catalytic antibodies. [10] Similar efficiencies were achieved by Wei and Hecht in de novo designed proteins that had not been screened or selected for esterase activity.…”

Section: Introductionmentioning

confidence: 99%

“…[16] Through the combination of the substrate design with enzyme engineering, a functional enzyme in which a catalytic His-His motif is grafted from a previously described peptide catalyst into the HCAII scaffold has been constructed. [9] Results…”

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confidence: 99%

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Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

et al. 2007

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A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum k(cat)/K(M) value of approximately 14 000 M(-1) s(-1), with a k(cat) value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.

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Section: Grafting Of a Helix Two-histidine Catalytic Site Into Hcaiimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

et al. 2007

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“…The studied reactions go through intermediates that are covalently linked to the catalyst, or have rate-limiting steps early in the reaction pathway, and bypass one aspect of biocatalysis that is especially difficult to mimic, namely the specific but differential binding of substrates, several intermediates and transition states by noncovalent forces. The acylated histidine side chain in His-mediated ester hydrolysis [32] and the imines in oxaloacetate decarboxylation [1,22] are illustrative examples of intermediates that are covalently bound to the catalyst. In the self-replication of helical peptides, catalysis has been achieved by bringing the reactants together for the first and rate-limiting bond-forming step and thus the binding of subsequent intermediates along the reaction pathway did not affect the catalytic efficiency.…”

Section: Introductionmentioning

confidence: 99%

Noncovalent Binding of a Reaction Intermediate by a Designed Helix‐Loop‐Helix Motif—Implications for Catalyst Design

Allert

Baltzer

2003

ChemBioChem

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In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.

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“…We thus set out to create miniproteins that would adopt a defined oligomeric state. Miniprotein models have been used to investigate biological processes and to model larger proteins through the incorporation of catalytic or other functionality (9)(10)(11). Oligomeric miniproteins serve as minimal model systems for quaternary structure formation.…”

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X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

Ali

Peisach

Allen

et al. 2004

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

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The x-ray crystal structure of an oligomeric miniprotein has been determined to a 1.2-Å resolution by means of multiwavelength anomalous diffraction phasing with selenomethionine analogs that retain the biophysical characteristics of the native peptide. Peptide 1, comprising ␣ and ␤ secondary structure elements with only 21 aa per monomer, associates as a discrete tetramer. The peptide adopts a previously uncharacterized quaternary structure in which ␣ and ␤ components interact to form a tightly packed and well defined hydrophobic core. The structure provides insight into the origins of the unusual thermal stability of the oligomer. The miniprotein shares many characteristics of larger proteins, including cooperative folding, lack of 1-anilino-8-naphthalene sulfonate binding, and limited deuterium exchange, and possesses a buried surface area typical of native proteins. O ligomerization is a fundamental strategy for generating protein structural and functional complexity in nature. Many large proteins are believed to have arisen from oligomeric precursors that, by means of gene duplication and fusion, have become one encoded protein (1). Some examples are the ribosome anti-association factor eIF6 (2), the periplasmic binding proteins (3, 4), and the eightfold ␤͞␣ barrel (5). Functionally, many proteins (6) are inactive in the monomeric state but are active as a dimer or higher-order oligomer; examples include GCN4 (7) and MCP-1 (8).We are interested in studying the structural and functional advantages conferred by protein oligomerization. We thus set out to create miniproteins that would adopt a defined oligomeric state. Miniprotein models have been used to investigate biological processes and to model larger proteins through the incorporation of catalytic or other functionality (9-11). Oligomeric miniproteins serve as minimal model systems for quaternary structure formation. Moreover, they constitute platforms for probing whether more complex oligomeric structures might ultimately support function.The strategy for oligomerization was inspired by ''domain swapping,'' or the exchange of association partners, an important evolutionary mechanism for protein oligomerization (12)(13)(14)(15). This strategy has previously been used for the design of a coiled-coil domain-swapped dimer (16). The prototypic ␤␤␣ (BBA) motif BBA5 (Fig. 1) was chosen as the building block because it represents a discretely folded and structured miniprotein motif (17,18). This motif includes interactions between secondary structural elements, thus presenting an opportunity to favor similar interactions between monomers within an oligomer. Because it has been reported that shortening the linker between domains favors domain-swapped oligomers (19, 20), we introduced a bias toward oligomerization in the BBA motif by varying the length of the 3-aa hinge region between secondary structural elements (21). Our approach for the discovery of homooligomers of peptides with ␤␤␣ supersecondary structure was based on a complementary design and screening ...

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Cooperative Nucleophilic and General-Acid Catalysis by the HisH⁺−His Pair and Arginine Transition State Binding in Catalysis of Ester Hydrolysis Reactions by Designed Helix−Loop−Helix Motifs

Cited by 72 publications

References 11 publications

Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

Noncovalent Binding of a Reaction Intermediate by a Designed Helix‐Loop‐Helix Motif—Implications for Catalyst Design

X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

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Cooperative Nucleophilic and General-Acid Catalysis by the HisH+−His Pair and Arginine Transition State Binding in Catalysis of Ester Hydrolysis Reactions by Designed Helix−Loop−Helix Motifs

Cited by 72 publications

References 11 publications

Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

Combined Enzyme and Substrate Design: Grafting of a Cooperative Two‐Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate

Noncovalent Binding of a Reaction Intermediate by a Designed Helix‐Loop‐Helix Motif—Implications for Catalyst Design

X-ray structure analysis of a designed oligomeric miniprotein reveals a discrete quaternary architecture

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Cooperative Nucleophilic and General-Acid Catalysis by the HisH⁺−His Pair and Arginine Transition State Binding in Catalysis of Ester Hydrolysis Reactions by Designed Helix−Loop−Helix Motifs