Directed Mutagenesis of Dihydrofolate Reductase

Villafranca, J. E.; Howell, Elizabeth E.; Voet, Donald; Strobel, Marjorie C.; Ogden, Richard C.; Abelson, John; Kraut, Joseph

doi:10.1126/science.6356360

Cited by 216 publications

(98 citation statements)

References 31 publications

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“…Kraut and co-workers observed that its replacement with Asn led to a fall in apparent overall catalytic competence to 0.1 % of that of the wild type enzyme. (Villafranca et al, 1983), and further studies have confirmed and extended this observation . Crystal structures for the wild type, Asp-27 --Asn, and Asp-27 -+ Ser variants of dihydrofolate reductase were compared at 0.19 nm resolution, and the only apparent structural differences noted, apart from the side chain atoms at the substitution site, were the positions of internally bound water molecules.…”

Section: Al-antitrypsinsupporting

confidence: 54%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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Section: Al-antitrypsinsupporting

confidence: 54%

Protein engineering. The design, synthesis and characterization of factitious proteins

Shaw

1987

Biochemical Journal

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“…However, oligonucleotide or site-directed mutagenesis offers an alternate and much more specific method for testing the role of any amino acid residue by selectively altering DNA codon sequences using in vitro recombinant DNA techniques. While the use of site-directed mutagenesis to study enzyme mechanisms is a field still in its infancy, it is clear from those few examples in the literature (13)(14)(15)(16) that such an approach provides a powerful tool for probing the details of enzymic catalysis. Therefore, we were encouraged to test the predicted role of the oxyanion hole in the subtilisin-catalyzed cleavage of a specific peptide substrate by changing Asn-155 to leucine.…”

Section: <C-nhmentioning

confidence: 99%

Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

Bryan¹,

Pantoliano²,

Quill³

et al. 1986

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

242

156

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Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtflisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The variant was found to have an unaltered K. but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.

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“…Oligonucleotide-directed mutagenesis has become a useful means for investigating the structure and function ofproteins (12). We have used the mutagenesis protocol to examine the role of individual amino acids in the reaction mechanism of the poliovirus protease.…”

mentioning

confidence: 99%

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

Ivanoff¹,

Towatari²,

Ray³

et al. 1986

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

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We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotidedirected mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histide-161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity.Poliovirus, a small RNA virus, is a member of the picornavirus family, which includes rhinovirus, encephalomyocarditis virus, and foot and mouth disease virus. During the viral life cycle, the genomic RNA is initially translated into a >200-kDa polyprotein, which, while nascent, is cleaved to a series of polypeptide intermediates, termed P1, P2, and P3. Further processing yields the majority of mature viral products (structural and nonstructural) and is dependent on a viral protease(s). The poliovirus P3 intermediate consists of three nonstructural proteins-the genome-linked virion protein (VPg), the protease (3C), and the replicase (3D)-and two proteins of unknown function (3C' and 3D') (1, 2). Hanecak et al. (3) have demonstrated that the 3C region of the poliovirus genome represents the core sequence responsible for the proteolytic processing (at Gln-Gly) of the precursor polypeptides. In addition, the poliovirus 3C protease has been reported to be autocatalytic (4), in agreement with results for encephalomyocarditis virus (5) and foot and mouth disease virus (6).The enzymatic mechanism of these viral proteases has not been well characterized. Inhibitors such as iodoacetamide, N-ethyl maleimide, and para-chloromercuribenzoate have implicated a cysteine residue in the active site (7,8). However, diisopropylphosphofluoridate, an inhibitor of serine proteases, has also been reported as an effective inhibitor of poliovirus protein processing (9). A comparative study (10) of the derived protease proteins from several picornaviruses as well as the related plant virus cowpea mosaic virus (11) revealed one region of significant homology located in the carboxyl third ofthe protein. Within this region a cysteine and a histidine residue have been strictly conserved. These observations suggest that the picornavirus proteases most probably belong to the class of cysteine proteases.To obtain sufficient quantities of the protease proteins for study, we have engineered a cDNA clone of the poliovirus 3C-3D region into an Escherichia coli expression vector, which produced the protease polypeptides in high yield...

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Directed Mutagenesis of Dihydrofolate Reductase

Cited by 216 publications

References 31 publications

Protein engineering. The design, synthesis and characterization of factitious proteins

Protein engineering. The design, synthesis and characterization of factitious proteins

Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli.

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