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D Hilvert

Publications and source records attributed to D Hilvert.

At least 37 records · Page 2Linked to original sources
PubMed1997

3D structural information as a guide to protein engineering using genetic selection.

A great variety of protein systems have been investigated in the past year using structure-guided evolutionary strategies. On the basis of available 3D structural information, critical regions of proteins have been targeted for randomizing mutagenesis and active variants of the corresponding genes have been selected. These approaches help characterize structural and mechanistic features of proteins and have important implications for design.

Binding Sites↗
PubMed1996

Selection of linkers for a catalytic single-chain antibody using phage display technology.

Phage display has been evaluated as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. Preliminary experiments with a conventional linker failed to yield a functional single-chain version of a catalytic antibody with chorismate mutase activity. A random linker library was therefore constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire ( approximately 5 x 10(6) different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers. There are apparently many viable solutions to the problem of linking individual VH and VL domains, but subtle differences in sequence dramatically influence the production, stability, and recognition properties of the scFv. The success of these experiments suggests that phage display will be generally useful for identifying peptide sequences for covalently linking any two protein domains.

Amino Acid Sequence↗
PubMed1996

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

Amino Acid Sequence↗
PubMed1996

Hydrolytic antibodies: variations on a theme.

Comparison of four independently-derived hydrolytic antibodies reveals striking similarities in their active sites. A common structural motif appears to be induced when the immune system is challenged with antigens containing aryl phosphonate and phosphonamidate groups, and key variations on this 'theme' must account for the observed differences in catalytic efficacy and mechanism. The limited structural repertoire accessed through standard immunization procedures suggests that new approaches may be needed to produce antibody catalysis with enzyme-like efficiencies.

Antibodies, Catalytic↗
PubMed1996

Antibody catalysis via strategic use of haptenic charge.

General acid-base catalysis contributes substantially to the efficacy of many enzymes. Similar effects can be exploited in antibody catalysis by taking advantage of charge complementarity between immunoglobulin and hapten (the template used to induce the antibody) to elicit functional groups in the combining site. This strategy has proved useful in the catalysis of a diverse set of chemical transformations, including elimination reactions. Provided that hapten design is optimized and the immune response is screened extensively, the efficiency of the resulting antibody catalysts can rival that of analogous natural enzymes.

Alkenes↗
PubMed1995

Nonessential active site residues modulate selenosubtilisin's kinetic mechanism.

Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the unmodified protease, selenosubtilisins derived from distantly related subtilisin templates exhibit significantly different kinetic properties. Selenosubtilisin BPN' not only is less active than the previously studied Carlsberg selenoenzyme but exhibits sequential rather than ping-pong kinetics, indicating the formation of a ternary complex between enzyme, thiol, and peroxide prior to product release. Experiments with subtilisin E and the BPN' Y217L variant show that the observed differences in kinetic mechanism and chemical efficiency can be attributed largely to amino acid substitutions in the enzyme's S1 and S1' binding sites, respectively. These contributions appear to be roughly additive, and a BPN' triple mutant (E156S/G169A/Y217L) has properties that closely approximate those of selenosubtilisin Carlsberg. The kinetic mechanism of selenosubtilisin can thus be controlled by limited mutagenesis of several active site residues not directly involved in the redox chemistry.

Binding Sites↗
PubMed1995

Large rate accelerations in antibody catalysis by strategic use of haptenic charge.

General acid-base catalysis contributes substantially to the efficacy of many enzymes, enabling an impressive array of eliminations, isomerizations, racemizations, hydrolyses and carbon-carbon bond-forming reactions to be carried out with high rates and selectivities. The fundamental challenge of exploiting similar effects in designed catalysts such as catalytic antibodies is that of correctly positioning the catalytic groups in an appropriate active-site microenvironment. Charge complementarity between antibody and hapten (the template used to induce an antibody) has been used successfully in a number of instances to elicit acids and bases within immunoglobulin combining sites, but the activities of the catalysts obtained by this strategy are generally considerably lower than those of natural enzymes. Here we report that by optimizing hapten design and efficiently screening the immune response, antibodies can be obtained that act effectively as general base catalysts. Thus a cationic hapten correctly mimicking the transition-state geometry of all reacting bonds and bearing little resemblance to the reaction product has yielded carboxylate-containing antibodies that catalyse an E2 elimination with more than 10(3) turnovers per active site and rate accelerations of greater than 10(8). These results demonstrate that very large effects can be achieved by strategic use of haptenic charge.

Antibodies, Catalytic↗
PubMed1994

Enrichment for RNA molecules that bind a Diels-Alder transition state analog.

RNA molecules that bind a transition state analog for a Diels-Alder reaction (Kd = 0.35 +/- 0.05 mM) were isolated from a starting pool of approximately 10(14) sequences by affinity chromatography. After the initial rise and plateau of the amount of RNA that eluted with soluble analog, a step gradient elution was used to further enrich the pool for sequences with higher affinities for the target. To our knowledge, the isolation of RNA molecules that bind either a nonplanar or a hydrophobic ligand has not been reported previously. A conserved nucleotide sequence and secondary structure present in many of the RNA molecules are necessary but not sufficient for binding the analog. No catalysts of the targeted Diels-Alder reaction were found among the binders. The absence of catalysis contrasts with previous successful experiments with antibodies and suggests that other strategies may be needed to identify oligonucleotides with diverse catalytic activities.

Base Sequence↗
PubMed1994

Routes to catalysis: structure of a catalytic antibody and comparison with its natural counterpart.

The three-dimensional structure of a catalytic antibody (1F7) with chorismate mutase activity has been determined to 3.0 A resolution as a complex with a transition state analog. The structural data suggest that the antibody stabilizes the same conformationally restricted pericyclic transition state as occurs in the uncatalyzed reaction. Overall shape and charge complementarity between the combining site and the transition state analog dictate preferential binding of the correct substrate enantiomer in a conformation appropriate for reaction. Comparison with the structure of a chorismate mutase enzyme indicates an overall similarity between the catalytic mechanism employed by the two proteins. Differences in the number of specific interactions available for restricting the rotational degrees of freedom in the transition state, and the lack of multiple electrostatic interactions that might stabilize charge separation in this highly polarized metastable species, are likely to account for the observed 10(4) times lower activity of the antibody relative to that of the natural enzymes that catalyze this reaction. The structure of the 1F7 Fab'-hapten complex provides confirmation that the properties of an antibody catalyst faithfully reflect the design of the transition state analog.

Antibodies, Catalytic↗
PubMed1994

Crystallization and preliminary structural studies of a chorismate mutase catalytic antibody complexed with a transition state analog.

The Fab' fragment of a catalytic antibody with chorismate mutase activity has been crystallized as a complex with the transition-state analog hapten. The complex was crystallized by the vapor diffusion method using ammonium sulfate as the precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 37.1 A, b = 63.3 A, c = 178.5 A, and there is one Fab' molecule per asymmetric unit. The crystals diffract X-rays to at least 3.0 A and are suitable for X-ray crystallographic studies.

Animals↗
PubMed1994

Chemical synthesis of proteins.

Powerful new enzymatic and chemical methods for coupling unprotected peptide fragments are making the assembly of large synthetic proteins possible. By allowing the use of novel backbones and the incorporation of multiple unnatural amino acids at specific sites, these methods promise large expansion of the repertoire of protein molecules accessible to research.

Proteins↗
PubMed1993

Peroxide dependence of the semisynthetic enzyme selenosubtilisin.

Selenosubtilisin, a semisynthetic enzyme produced by chemical modification of subtilisin's catalytic serine, mimics the antioxidant enzyme glutathione peroxidase, catalyzing the reduction of hydroperoxides by 3-carboxy-4-nitrobenzenethiol. In analogy with the natural peroxidase, a variety of hydroperoxides are accepted as substrates for the semisynthetic enzyme, whereas the dialkyl compound tert-butyl peroxide is not. Kinetic investigations reveal that kmax is dependent upon the nature of the hydroperoxide, indicating that peroxide-mediated oxidation of the enzymic selenolate is at least partially rate-limiting. Experiments with the radical trap 2,6-di-tert-butyl-4-methylphenol suggest that, while the nonenzymic reaction between tert-butyl hydroperoxide and thiol involves free radicals, the same reaction catalyzed by selenosubtilisin does not. The studies described here support the enzyme's proposed ping-pong mechanism and are consistent with previous mechanistic observations.

Catalysis↗
PubMed1993

Binding of a high-energy substrate conformer in antibody catalysis.

Enzymes can substantially increase the probability of a reaction by exploiting binding energy to preorganize their substrates into reactive conformations. Similar effects are likely to be important in a wide variety of designed catalysts, including catalytic antibodies. Transferred nuclear Overhauser effects have been used here to investigate how an antibody possessing chorismate mutase activity binds its flexible substrate molecule chorismate. The conversion of chorismate to prephenate by way of a Claisen rearrangement requires the substrate to adopt an energetically disfavored diaxial conformation in which the enolpyruvyl side chain is positioned over the six-membered ring. The antibody, which was elicited by a conformationally restricted transition state analog for this reaction, appears to bind this high-energy substrate conformer preferentially, as judged by diagnostic intramolecular transferred nuclear Overhauser effects. Inhibitor studies with the transition state analog confirm that preorganization takes place exclusively at the antibody active site. These results thus provide strong physical evidence for a direct relationship between the properties of a catalytic antibody and the structure of the transition state analog originally used to elicit the immune response.

Antibodies, Monoclonal↗
PubMed1993

Crystal structure of selenosubtilisin at 2.0-A resolution.

The three-dimensional structure of selenosubtilisin, an artificial selenoenzyme, has been solved at 2.0-A resolution by the method of molecular replacement. Selenosubtilisin is a chemical derivative of the bacterial serine protease subtilisin in which the catalytically essential serine residue has been replaced with a selenocysteine. Its unique hydrolytic and redox properties reflect the intrinsic chemical reactivity of the selenium prosthetic group. Structural analysis of the modified protein reveals that the selenium moiety is selectively incorporated into the side chain of residue 221 and confirms the seleninic acid oxidation state expected from treatment of the enzyme with hydrogen peroxide prior to crystallization. Although the seleninic acid replaces the essential nucleophile in the enzyme's catalytic triad and introduces a negative charge into the active site, the interaction between His64 and Asp32 is not altered by the modification. Hydrogen bonds from the oxygen atoms of the seleninic acid to His64 and to Asn155 in the oxyanion hole confine the prosthetic group to a single well-defined conformation within the active site. These interactions thus provide a structural basis for understanding the seleninic acid's unusually low pKa, the enzyme's relatively sluggish rate of reaction with thiols, and its much more efficient peroxidase activity. Aside from the active site region, the structure of the protein is essentially the same as that previously reported for native subtilisin Carlsberg, indicating the viability of chemical modification strategies for incorporating site-specific changes into the protein backbone. Comparison of the three-dimensional structures of selenosubtilisin and glutathione peroxidase, an important naturally occurring selenoenzyme, provides the means to evaluate how the function of the selenium prosthetic group varies with molecular context.

Binding Sites↗
PubMed1993

Kinetic studies on the peroxidase activity of selenosubtilisin.

Selenosubtilisin, a semisynthetic selenoenzyme produced by chemical modification of the serine protease subtilisin, acts as a mimic of glutathione peroxidase, catalyzing the reduction of tert-butyl hydroperoxide by 3-carboxy-4-nitrobenzenethiol. To clarify the mechanism of action of this catalyst, detailed kinetic studies have been carried out. Thiol-mediated reduction converts the seleninic acid form of selenosubtilisin (ESeO2H) into a selenenyl sulfide (ESeSAr). Investigations into the reduction of ESeO2H by the aromatic thiol revealed saturation kinetics and were consistent with a significant lowering of the pKa of the seleninic acid in the enzyme active site. While the reduction of ESeO2H was slow compared with a simple model system, the reduced selenoenzyme (ESeSAr) exhibited a much greater peroxidase activity than model compounds. The enzymic selenocysteine residue was shown to be crucial for this activity, and ping-pong kinetics were observed. A catalytic cycle involving interconversion of the ESeSAr, ESeH, and ESeOH forms of the enzyme has been proposed that is consistent with all the available data. The pH-rate profile for the peroxidase activity indicates the involvement of the active site histidine (His64) in the rate-determining step, which these investigations suggest is attack of ArS- on ESeSAr. The results presented here correlated well with crystallographic and spectroscopic data and provide more detailed information about crucial interactions within the active site of selenosubtilisin.

Binding Sites↗
PubMed1993

1H NMR spectroscopic studies of selenosubtilisin.

Anomalously low-field signals in 1H NMR spectra of serine proteases provide valuable information on the protonation state of the catalytic histidine residue. We have examined the pH dependence of the deshielded protons of three different oxidation states of selenosubtilisin, a semisynthetic selenoenzyme with significant peroxidase activity, in order to evaluate the influence of the selenium prosthetic group on the hydrogen-bonding network in the modified active site. In the spectra of the anionic seleninate and selenolate derivatives, two resonances were observed at 18.0 and 15.5/14.0 ppm, assigned respectively to the N delta 1 and N epsilon 2 protons of protonated His64. These signals were apparent from pH 4 to above pH 10, indicating that the negatively charged prosthetic group increases the stability of the imidazolium dramatically, raising its pKa by at least 3-4 pH units. In contrast, a neutral selenenyl sulfide species exhibits no deshielded proton signals at 18 ppm at any pH but has a weak signal at 14.1 ppm above pH 7 which was assigned to the N delta 1 imidazole proton of neutral His64. While the pKa of His64 appears normal (approximately 7) in this derivative, the selenenyl sulfide substitution may alter the orientation of the imidazole ring within the active site for steric reasons. Together with data on the influence of pH on peroxidase activity, these results suggest that selenosubtilisin's His64 acts as a general acid facilitating the reduction of the selenenyl sulfide to selenolate by thiols.

Binding Sites↗
PubMed1991

In vivo catalysis of a metabolically essential reaction by an antibody.

We have established a growth selection requirement for a catalytic antibody with modest chorismate mutase activity. Conversion of (-)-chorismate into prephenate is the key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. Strains of the yeast Saccharomyces cerevisiae containing an insertion mutation in the structural gene for the enzyme chorismate mutase (EC 5.4.99.5) require exogenous supplements of these two amino acids for efficient growth. Intracellular expression of the heterologous antibody catalyst in one such strain, identified by random mutagenesis and genetic selection, provides a substantial growth advantage under auxotrophic conditions; complementation was not observed with an unrelated esterolytic antibody. In addition to demonstrating that tailored immunoglobulin catalysts can carry out vital biochemical reactions in vivo, these experiments provide a powerful selection assay for identifying genetic changes within the antibody molecule itself that augment chemical efficiency.

Antibodies↗