Biomedical subjects

D R Corey

Publications and source records attributed to D R Corey.

14 recordsLinked to original sources

PubMed1996

Distinct mechanisms contribute to stringent substrate specificity of tissue-type plasminogen activator.

Tissue-type plasminogen activator (t-PA) has evolved to optimize cleavage of plasminogen (Plg) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2 subsites of t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly selective cleavage of protein fusions. Surprisingly, t-PA cleaves this engineered protein substrate with a Km that is reduced 950-fold relative to the Km for hydrolysis of the same target sequence within a peptide. This reduction of Km suggests that binding is facilitated by interactions between protein substrate and protease that are distant from the P4-P2' residues. We use this kinetic data to derive a model in which several distinct mechanisms contribute to the remarkable specificity of t-PA.

Amino Acid Sequence↗

PubMed1996

Telomerase: anti-cancer target or just a fascinating enzyme?

Telomerase activity is upregulated in most types of malignant tumor. Highly selective small molecule inhibitors will be needed to understand the biological basis for this observation and to determine if telomerase is a viable target for chemotherapy.

Enzyme Inhibitors↗

PubMed1995

Trypsin specificity increased through substrate-assisted catalysis.

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.

Amino Acid Sequence↗

PubMed1995

Origins of the specificity of tissue-type plasminogen activator.

The role of subsite interactions in defining the stringent substrate specificity of tissue-type plasminogen activator (t-PA) has been examined by using an fd phage library that displayed random hexapeptide sequences and contained 2 x 10(8) independent recombinants. Forty-four individual hexapeptides were isolated and identified as improved substrates for t-PA. A peptide containing one of the selected amino acid sequences was cleaved by t-PA 5300 times more efficiently than a peptide that contained the primary sequence of the actual cleavage site in plasminogen. These results suggest that small peptides can mimic determinants that mediate specific proteolysis, emphasize the importance of subsite interactions in determining protease specificity, and have important implications for the evolution of protease cascades.

Amino Acid Sequence↗

PubMed1995

Accelerated hybridization of oligonucleotides to duplex DNA.

We report two strategies for accelerating the hybridization of oligonucleotides to DNA. We demonstrate that oligodeoxyribonucleotides and peptide nucleic acid oligomers hybridize to inverted repeats within duplex DNA by D-loop formation. Oligonucleotides and duplex template form an active complex, which can be recognized by T7 DNA polymerase to prime polymerization. Quantitation of polymerization products allowed the rate of hybridization to be estimated, and peptide nucleic acid oligomers and oligonucleotide-protein adducts anneal with association constants 500- and 12,000-fold greater, respectively, than the analogous unmodified oligonucleotides. Together, these results indicate that sequences within duplex DNA can be targeted by Watson-Crick base pairing and that chemical modifications can dramatically enhance the rate of strand association. These findings should facilitate targeting of oligomers for priming DNA polymerization, the detection of diagnostic sequences, and the disruption of gene expression. The observed acceleration of hybridization may offer a new perspective on the ability of RecA or other proteins to accelerate strand invasion.

Base Sequence↗

PubMed1995

Substrate specificity of tissue type plasminogen activator. Characterization of the fibrin independent specificity of t-PA for plasminogen.

Tissue-type plasminogen activator (t-PA) is a remarkably specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg560-Val561) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction between fibrin, t-PA and plasminogen which reduces the Km of t-PA for plasminogen by a factor of 440. However, even in the absence of fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does trypsin, a homologous serine protease. We have measured the extent of the specificity of t-PA for plasminogen by assaying t-PA and trypsin toward substrates modeled after the peptide sequence in plasminogen surrounding Arg560-Val561. Surprisingly, t-PA hydrolyzes these substrates with kcat/Km values which are 28,000-210,000-fold lower than those obtained using trypsin. Both the high activity toward plasminogen and the low activity toward peptides are also exhibited by the isolated protease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specific for recognition of one or more structural features displayed by native plasminogen.

Amino Acid Sequence↗

PubMed1995

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA.

A convenient small scale synthesis has been developed for obtaining peptide nucleic acid oligomers (PNAs). PNAs have been conjugated to a protein, staphylococcal nuclease, through disulfide exchange between a cysteine at the 3'-(carboxy) end of the PNA and an introduced cysteine on the surface of the nuclease. Site specific DNA cleavage by the attached nuclease has been used to examine the Watson-Crick hybridization of the PNAs to duplex DNA. Substantial affinity cleavage occurred when target sites contained inverted repeats which have the potential to form non B-DNA structures such as cruciforms. No affinity cleavage was observed at a site lacking apparent potential for non B-DNA structures. These results indicate that the Watson-Crick hybridization of PNAs to duplex DNA by strand displacement is favored by the presence of potential alternative secondary structures within the target sequence.

Base Sequence↗

PubMed1994

Cyclic peptides as proteases: a reevaluation.

A recent report [Atassi, M. Z. and Manshouri, T. (1993) Proc. Natl. Acad. Sci. USA 90, 8282-8286] described the design and synthesis of two 29-amino acid cyclic peptides that were reported to hydrolyze both ester and amide bonds with chymotrypsin-like or trypsin-like specificity. We have synthesized the trypsin-mimic peptide (TrPepz) and detect no activity toward either ester or peptide substrates. The same result was independently obtained by Wells et al. [Wells, J. A., Fairbrother, W. J., Otlewski, J., Laskowski, M., Jr., & Burnier, J. (1994) Proc. Natl. Acad. Sci. USA 91, 4110-4114.] Additionally, we found that Atassi and Manshouri failed to obtain accurate kinetic constants for trypsin- and chymotrypsin-catalyzed ester hydrolysis because the high concentrations of trypsin and chymotrypsin that they report using would have prevented evaluation of initial rates. These findings are incompatible with the claims, reported by Atassi and Manshouri, that TrPepz has trypsin-like activity.

Amino Acid Sequence↗

PubMed1993

Trypsin display on the surface of bacteriophage.

The gene III and VIII-encoded coat proteins (pIII and pVIII) from bacteriophage M13 have been fused to the C terminus of the serine protease, trypsin (Tsn). The genes encoding the fusions were then inserted directly into M13mp18 to create vectors which expressed both the Tsn-coat protein hybrids and the wild-type (wt) coat proteins. Immunoblot analysis confirmed that the bacteriophage express Tsn on their surface. Isolated fusion phage possess kinetic parameters which approximate those of the wt enzyme. An endogenous Escherichia coli protease inhibitor, ecotin, copurifies with the Tsn phage. Immobilized ecotin can be used to selectively bind bacteriophage which express Tsn::pIII fusion proteins.

Animals↗

PubMed1990

Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation.

A Lys-84----Cys mutant staphylococcal nuclease was selectively linked to the 5' and/or 3' terminus of a thiol-containing polypyrimidine oligonucleotide via a disulfide bond. The oligonucleotide-staphylococcal nuclease adduct is capable of binding to a homopurine-homopyrimidine region of Watson-Crick duplex DNA by the formation of a triple-helical structure. Upon the addition of Ca2+, the nuclease cleaves DNA at sites adjacent to the homopurine tract. Specific double-strand cleavage occurred predominantly at A + T-rich sites to the 5' side of the homopurine tract for both the 5'-derivatized and the 5',3'-diderivatized nucleases; the 3'-derivatized nuclease gave no cleavage. The cleavage pattern is asymmetric and consists of multiple cleavage sites shifted to the 5' side on each strand, centered at the terminal base pair of the binding site. Microgram amounts of plasmid pDP20 DNA (4433 base pairs) containing a homopurine-homopyrimidine tract were selectively cleaved by a semisynthetic nuclease with greater than 75% efficiency at room temperature within 1 hr. Cleavage reaction conditions were optimized with respect to pH, temperature, reaction times, and reaction components. Semisynthetic nucleases of this type should provide a powerful tool in chromosomal DNA manipulations.

Base Sequence↗

PubMed1989

Generation of a catalytic sequence-specific hybrid DNase.

Hybrid nucleases consisting of an oligonucleotide fused to a unique site on the relatively nonspecific phosphodiesterase staphylococcal nuclease have been shown to sequence specifically cleave DNA. We have introduced mutations into the binding pocket of the nuclease which lower the kcat/Km of the enzyme. Hybrid nucleases generated from these mutants sequence selectively hydrolyze single-stranded DNA in a catalytic fashion, and under a much wider range of conditions than was previously possible. One such hybrid nuclease (Y113A, K116C) was able to site selectively cleave single-stranded M13mp7 DNA (7214 nt), primarily at one phosphodiester bond. Another hybrid nuclease (Y113A, L37A, K116C) catalyzed the hydrolysis of a 78-nt DNA substrate with a kcat of 1.2 min-1 and a Km of 120 nM. The effects of variations in the length and sequence of the oligonucleotide binding region were examined, as were changes in the length of the tether between the oligonucleotide and the enzyme. Cleavage specificity was also assayed as a function of substrate DNA primary and secondary structure and added poly(dA).

Base Sequence↗

PubMed1989

Introduction of a metal-dependent regulatory switch into an enzyme.

A cysteine has been introduced into the hydrophobic binding pocket of staphylococcal nuclease via oligonucleotide-directed mutagenesis. The L89C mutation does not significantly alter the catalytic activity or specificity of the nuclease yet provides a metal-dependent switch for regulating enzymatic activity. The L89C mutant can be inactivated by addition of mercuric or cupric salts and subsequently reactivated by addition of chelating agents. This work may provide a general strategy for regulating the catalytic activity of other enzymes or the binding affinity of proteins to DNA or other proteins.

Chelating Agents↗

PubMed1987

Generation of a hybrid sequence-specific single-stranded deoxyribonuclease.

The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys116 in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3'-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.

Amino Acid Sequence↗

PubMedDate not supplied

Strand invasion by oligonucleotide--nuclease conjugates.

Conjugates consisting of staphylococcal nuclease crosslinked to oligonucleotides hybridize to supercoiled duplex DNA by Watson--Crick base-pairing. Here we describe this strand invasion. Affinity cleavage by these conjugates provides a probe for the local topology of the DNA duplex and is most efficient at a target DNA sequence known to form a cruciform. Additional supercoiling of the substrate DNA increases selective cleavage at other sequences. Hybridization of the conjugate to duplex DNA is temperature dependent and is stable over time. Affinity cleavage is not substantially inhibited by a 200-fold excess of the analogous unmodified oligonucleotide, demonstrating that hybridization of the unmodified oligonucleotide must be less favored and that the nuclease is involved in substrate binding. Surprisingly, affinity cleavage is also not effectively inhibited by complementary oligonucleotides unless they contain an extended 5'-sequence capable of separate interactions with the nuclease domain of the conjugate. These results suggest that the oligonucleotide-nuclease conjugate prefers to hybridize to target sequences which will allow interactions with both the oligonucleotide and the nuclease domains. Affinity cleavage by oligonucleotide-nuclease conjugates provides general insights for the design of oligonucleotides and their conjugates for strand invasion and affords a convenient competition assay for their hybridization.

Amino Acid Sequence↗