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Biomedical subjects

R A Copeland

Publications and source records attributed to R A Copeland.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism.

Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

ADAM Proteins↗
PubMed1999

Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase.

CO2-capture methods have been used for assaying many decarboxylating enzymes including hydroxylation-coupled decarboxylation reactions. The traditional CO2-capture method involves performing the reaction in capped tubes and radiometric measurement of trapped 14CO2 by scintillation counting. In this report, a 14CO2-capture method in a 96-well microtiter plate format has been developed and a phosphor imaging system has been employed for sample measurement. The new assay method has been used successfully to assay aspartyl-beta-hydroxylase activity in microtiter plate format. The results obtained here compare favorably with those obtained from the traditional tube method. The method is sensitive, suitable for high throughput, and generally applicable to many CO2-releasing enzyme assays.

Carbon Dioxide↗
PubMed1999

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

ADAM Proteins↗
PubMed1999

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Crystallography, X-Ray↗
PubMed1999

The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

Animals↗
PubMed1998

MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.

In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.

Animals↗
PubMed1998

Identification of a novel inhibitor of mitogen-activated protein kinase kinase.

The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.

Animals↗
PubMed1998

Competitive inhibition of MAP kinase activation by a peptide representing the alpha C helix of ERK.

On the basis of the crystal structure of the MEK substrate ERK, we have synthesized a 15 amino acid peptide representing the alpha C helix of human ERK1. We find this peptide to be an inhibitor of ERK phosphorylation by its upstream activator MEK. Circular dichroic spectroscopy indicates that the peptide has little secondary structure in aqueous buffer, but can readily adopt an alpha-helical structure in aprotic solvent. Steady-state kinetic analysis indicates that the peptide serves as a competitive inhibitor of ERK binding to MEK, with a dissociation constant, Ki, of 0.84 microM. Together with ATP-competitive inhibitors of MEK, we have used this peptide to define the kinetic mechanism of MEK catalysis. These studies reveal that MEK operates through a bi-bi random-ordered sequential mechanism. The synthetic peptide inhibits also the phosphorylation of p38 and ERK by the upstream activator MKK3, but is at least 3-fold less potent as an inhibitor of SEK activation of JNK1. Interestingly, the peptide also showed some ability to inhibit ERK-mediated phosphorylation of myelin basic protein, but was inactive as an inhibitor of the unrelated kinases Raf, Abl, and PKA. These results imply that the alpha C helix is an important locus of interaction for the formation of a MEK-ERK complex. The alpha C helix cannot, however, be the sole determinant of activator selectivity among the MAP kinases. Molecules designed to target the alpha C helix binding pocket of MAP kinase activators may provide a novel means of inhibiting these signal transducers.

Amino Acid Sequence↗
PubMed1998

Kinetic effects due to nonspecific substrate-inhibitor interactions in enzymatic reactions.

Nonspecific protein binding is a commonly encountered problem with synthetic molecules designed as enzyme inhibitors. When the substrate for the enzymatic reaction is itself a protein, such nonspecific protein binding can also occur. Here, we demonstrate that this phenomenon can have a dramatic effect on the steady-state kinetic evaluation of such inhibitors.

Catalysis↗
PubMed1997

Histidine to alanine mutants of human dihydroorotate dehydrogenase. Identification of a brequinar-resistant mutant enzyme.

Dihydroorotate dehydrogenase (DHODase) is the rate-limiting enzyme of the mammalian de novo pyrimidine biosynthesis pathway, and is the molecular target of the antiproliferative, immunosuppressive compound brequinar sodium (BQR). We have shown previously that the activity of the recombinant human enzyme displays pH and diethylpyrocarbonate sensitivities that implicate a critical role for one or more histidine residues in catalysis [Copeland et al., Arch Biochem Biophys 323: 79-86, 1995.]. Here we report the results of alanine scanning mutagenesis for each of the 8 histidine residues of the recombinant human enzyme. In most cases, the replacement of histidine by alanine had little effect on the Km values of the two substrates, dihydroorotate and ubiquinone, or on the overall kcat of the enzymatic reaction. Replacement of H71, H129, and H364 by alanine, however, completely abolished enzymatic activity. The loss of activity for the H71A mutant was unexpected, since this residue is not conserved in the homologous rat enzyme; in the rodent enzyme this residue is an asparagine. Replacement of H71 by asparagine in the human enzyme led to a full recovery of enzymatic activity, indicating that a histidine is not required at this position. Replacement of H26 by alanine led to about a 10-fold reduction in catalytic activity relative to the wild-type enzyme, with no significant perturbation of the substrate Km values. This mutant was, however, at least 167-fold less sensitive to inhibition by the noncompetitive inhibitor BQR. While the wild-type and other mutant enzymes displayed IC50 values for BQR inhibition between 6 and 10 nM, the H26A mutant was inhibited less than 25% at concentrations of BQR as high as 150 nM. These data suggest that H26 plays an important role in BQR binding to the enzyme.

Alanine↗
PubMed1997

Detection of an Fe2+-protoporphyrin-IX intermediate during aspirin-treated prostaglandin H2 synthase II catalysis of arachidonic acid to 15-HETE.

Spectral intermediates associated with the dioxygenase and peroxidase activities of prostaglandin H2 (PGH2) synthase I and II were monitored by stopped-flow spectrometry. During reactions of PGH2 synthase I with arachidonic acid (AA) and ethyl hydrogen peroxide (EtOOH), compound I (Fe5+; formally (protoporphyrin-IX) x +Fe4+=O) and compound II (Fe4+; formally (protoporphyrin-IX)Fe4+=O) were detected. These intermediates were observed sooner with EtOOH (within 50 ms) than with AA (within 200 ms). Compound I and compound II were found to be kinetically competent with respect to AA-dependent O2 uptake. These findings are consistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. During reactions with PGH2 synthase II with AA, compound I and compound II were again observed within 200 ms and were kinetically competent to participate in dioxygenation. However, during reactions of PGH2 synthase II with EtOOH, compound I and compound II were detected much later (after 10 s). These findings would be inconsistent with a mechanism in which peroxidative cleavage precedes AA dioxygenation. When aspirin-treated PGH2 synthase II was reacted with EtOOH, a normal peroxidase cycle occurred with compound I and compound II formation occurring over 10 s. However, when aspirin-treated PGH2 synthase II was reacted with AA, a unique spectral intermediate with lambda(max) at 446 nm was detected within 3 ms and was strikingly similar to ferrous (Fe2+) protoporphyrin-IX. Aspirin-treated PGH2 synthase II was found to produce 15-HETE, and the appearance of the Fe2+ intermediate (within 3 ms) indicated that it was kinetically competent to participate in the 15-dioxygenation event. The detection of this Fe2+ intermediate and the slow formation of compound I and compound II observed with EtOOH in PGH2 synthase II suggest that peroxidative cleavage is not the initiating event in dioxygenation. Instead, it is proposed that the reduction of Fe3+ in heme to Fe2+ oxidizes a peroxide to yield an initiating peroxy radical. Since it is unlikely that 11- and 15-dioxygenation occurs via different mechanisms, our findings question mechanisms of catalysis in both PGH2 synthases.

Arachidonic Acid↗