Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structure of procarboxypeptidase A (PCPA) from porcine pancreas has been determined at 2 A resolution and refined to a crystallographic R-factor of 0.198, with a root-mean-square deviation from ideal values for bond lengths of 0.015 A and for angles of 2.1 degrees. It is compared with procarboxypeptidase B (PCPB) from the same tissue. The 94/95 residue activation segments of PCPA/PCPB have equivalent folds: an N-terminal globular region with an open sandwich antiparallel alpha/antiparallel beta topology, followed by an extended alpha-helical segment, the connection to the enzyme. Alignment of the secondary structures of the activation segments of PCPA and PCPB (residues A1 to A99) indicates a two residue insertion between residues A34 and A35 and a C-terminal helix that is two turns longer in PCPA compared to PCPB. A deletion is observed between residues A43 to A45, the region containing the short 3(10) helix that covers the active site in PCPB. The globular region (A4 to A80) shields the preformed active center of carboxypeptidase A (CPA), but none of the residues involved in catalysis makes direct contacts with the activation segment. In contrast, subsites S2, S3 and S4 of the enzyme, involved in the binding of peptidic substrates, are blocked by specific contacts with residues AspA36, TrpA38, ArgA47, AspA53 and GluA86 of the activation segment. It has been described that several residues of CPA exhibit different conformations in the free enzyme compared to when substrate is bound: Arg127, Arg145, Glu270 and Tyr248. In PCPA all of these residues are found in the "active" conformation, as if substrate were actually bound. The presence of a ligand, tentatively interpreted as a free amino acid (Val) in the active center could explain this fact. The connecting region (A80 to A99), the target for proteolytic activation, establishes fewer contacts with the enzyme in PCPA than in PCPB. The activation segment of PCPA (A4 to A99) remains bound to the enzyme after the first trypsin cleavage between ArgA99-Ala1 probably due to the stability conferred on it by the alpha-helix (alpha 3) of the connecting segment. These and other structural features may explain the differences in intrinsic activity and different rates or proteolytic activation of each zymogen.
J Mol Biol 1992 Mar 05
PMID:Three-dimensional structure of porcine pancreatic procarboxypeptidase A. A comparison of the A and B zymogens and their determinants for inhibition and activation. 154 96

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.
Mol Cell Biol 1992 Apr
PMID:Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes. 154 11

Protease injury of the bronchial epithelium may play an important role in the pathogenesis of many airway diseases including asthma and chronic bronchitis. We hypothesized that neutrophil elastase can cause significant injury to the bronchial epithelium leading to detachment of bronchial epithelial cells. This detachment might be prevented if elastase is released into the airway lumen and the bronchial epithelium forms a barrier preventing access to the basal attachment sites. To assess this, the detachment of bronchial epithelial cells by elastase from extracellular matrix was measured. An increase in the resistance to detachment with time in culture was observed. The resistance to detachment was confirmed in bronchial epithelial cells, which were grown to electrically resistant monolayers on millipore filters and exposed to trypsin and elastase applied to both the apical and basal surfaces. Significantly greater detachment occurred when the proteases were applied at the basal surface versus the apical surface. Injury to the bronchial epithelium may enhance the proteolytic effect on the epithelium by disrupting epithelial barrier function. This was tested by exposing bronchial epithelial cell cultures to cigarette smoke extract before exposure to trypsin and elastase. The detachment of the bronchial epithelial cells exposed on the apical surface was increased greatly after smoke exposure. These data suggest that an intact bronchial epithelium can act as a barrier against proteolytic injury. Such a mechanism might protect the airway epithelium during intraluminal inflammation, and, if defective, might potentiate cigarette smoke-induced airway injury.
Am J Respir Cell Mol Biol 1992 Apr
PMID:Observations of development of resistance to detachment of cultured bovine bronchial epithelial cells in response to protease treatment. 155 Jun 86

We have investigated the effects of ligand and DNA binding on the structure of the oestrogen receptor by performing limited proteolysis and analysing DNA binding activity by gel shift analysis. The effects of oestradiol, 4-hydroxytamoxifen and ICI 164,384 have been examined and we have found that despite differences in the DNA binding activity or relative mobility of the receptor-DNA complex we were unable to detect differences in the cleavage pattern produced by trypsin, chymotrypsin, Staphylococcus aureus V8, papain or elastase. Inhibition of DNA binding by ICI 164,384 was lost in receptor fragments that lacked the hormone binding domain. In contrast to the full-length receptor, proteolytic fragments produced by chymotrypsin differed in their ability to bind to an oestrogen response element (ERE) vs a thyroid response element (TRE). Evidence is presented that this difference can be accounted for by the inability of fragments lacking the hormone binding domain to dimerise on a TRE.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Effect of ligand binding and DNA binding on the structure of the mouse oestrogen receptor. 156 7

A new computer program is described, which positions small molecules into clefts of protein structures (e.g. an active site of an enzyme) in such a way that hydrogen bonds can be formed with the enzyme and hydrophobic pockets are filled with hydrophobic groups. The program works in three steps. First it calculates interaction sites, which are discrete positions in space suitable to form hydrogen bonds or to fill a hydrophobic pocket. The interaction sites are derived from distributions of nonbonded contacts generated by a search through the Cambridge Structural Database. An alternative route to generate the interaction sites is the use of rules. The second step is the fit of molecular fragments onto the interaction sites. Currently we use a library of 600 fragments for the fitting. The final step in the present program is the connection of some or all of the fitted fragments to a single molecule. This is done by bridge fragments. Applications are presented for the crystal packing of benzoic acid and the enzymes dihydrofolate reductase and trypsin.
J Comput Aided Mol Des 1992 Feb
PMID:The computer program LUDI: a new method for the de novo design of enzyme inhibitors. 158 40

The accessible surface, described by Lee and Richards (the L&R surface: J. Mol. Biol. 1971, 55, 379), has remarkably useful properties for displaying ligand-protein interactions. The surface is placed one van der Waals radius plus one probe radius away from the protein atoms. The ligands are displayed in skeletal form. With a suitable probe radius, those parts of the ligand in good van der Waals contact with the protein binding site are found superimposed on the L&R surface. Display of the surface using parallel contours therefore provides a very powerful guide for interactive drug design because only ligand atoms lying on or close to the surface are in low-energy contact. The ability of the surface to accurately display steric complementarity between ligands and proteins was optimized using data from small molecule crystal structures. The possibility of displaying the chemical specificity of the binding site was also investigated. The surface can be colored to give precise information about chemical specificity. Electrostatic potential, electrostatic gradient, and distance to hydrogen-bonding groups were tested as methods of displaying chemical specificity. The ability of these methods to describe the complementarity actually observed in the interior of proteins was compared. High-resolution crystal data for ribonuclease and trypsin was used. The environment surrounding extended peptide chains in the protein was treated as a virtual binding site. The peptide chain served as a virtual ligand. This large sample of experimental data was used to measure the correlation between type of ligand atom and the calculated property of the nearest binding site surface. The best correlation was obtained using hydrogen-bonding properties of the binding site. Using this parameter the surface could be divided into three separate zones representing the hydrophobic, hydrogen-bond-acceptor, and hydrogen-bond-donor properties of the binding site. The percentage of hydrophobic ligand atoms found to lie closest to the hydrophobic protein surface was 91%. The equivalent scores for ligand hydrogen-acceptor atoms and hydrogen-donor atoms found at the corresponding complementarity zone were 94% and 91%. The surface zones can be readily displayed using three colors. To test the method on real ligand/binding site interactions, nine thermolysin-inhibitor complexes of known structure were evaluated using the parameters and criteria derived from the protein-packing study and a correlation between complementary contacts and logarithm of potency was obtained which had an r2 of 0.99.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Definition and display of steric, hydrophobic, and hydrogen-bonding properties of ligand binding sites in proteins using Lee and Richards accessible surface: validation of a high-resolution graphical tool for drug design. 158 50

The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid beta precursor protein (beta APP). It is known that beta APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present beta APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in beta APP have an evolutionarily close relationship with the inter-alpha-trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4+ lymphocytes.
J Mol Evol 1992 Jun
PMID:Evolutionary origin of a Kunitz-type trypsin inhibitor domain inserted in the amyloid beta precursor protein of Alzheimer's disease. 159 45

The binding surface of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in contact with alpha-chymotrypsin has been identified by measurement of the change in amide hydrogen-exchange rates between free and chymotrypsin-bound inhibitor. Exchange measurements were made for the enzyme-bound form of the inhibitor at pH 7.3, 25 degrees C using fast-flow affinity chromatography and direct measurement of exchange rates in the protein complex from one-dimensional and two-dimensional nuclear magnetic resonance spectra. The interface is characterized by a broad surface of contact involving residues 39 through 48 of the anti-chymotryptic domain beta-hairpin as well as residues 32, 33 and 37 in the anti-chymotryptic domain loop of the inhibitor. A number of residues in the anti-tryptic domain of the protein also have an altered exchange rate, suggesting that there are changes in the protein conformation upon binding to chymotrypsin. These changes in amide exchange behavior are discussed in light of a model of the complex based on the X-ray crystallographic structure of turkey ovomucoid inhibitor third domain bound to a alpha-chymotrypsin, and the structure of free Bowman-Birk inhibitor determined in solution by two-dimensional nuclear magnetic resonance spectroscopy. The chymotrypsin-binding loop of Bowman-Birk inhibitor in the model is remarkably similar to the binding loop conformation in crystal structures of enzyme-bound polypeptide chymotrypsin inhibitor-I from potatoes, turkey ovomucoid inhibitor third domain, and chymotrypsin inhibitor-II from barley seeds.
J Mol Biol 1992 Jun 05
PMID:Identification of a protein-binding surface by differential amide hydrogen-exchange measurements. Application to Bowman-Birk serine-protease inhibitor. 160 87

Cytolytic granules purified from natural killer lymphocytes (NK) contain a pore-forming protein (perforin) and a number of serine proteases. When these proteases are inhibited by serine protease-specific isocoumarin reagents the serine proteases are inactivated and the cytolytic activity of the granules is decreased. Paradoxically, it has been found that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) frequently cannot block killing even though it inhibits many of the serine proteases. At the same time it has been reported that "purified" perforin alone can lyze cells. To address these inconsistencies we first compared the ability of PMSF and four new sulfonyl fluoride serine protease inhibitors to inhibit proteases and cell lysis. We determined the effects on lysis and the second order inhibition rate constants for five granule protease activities: ly-tryptase, ly-chymase, Met-ase (methionine cleaving), Ser-ase (serine cleaving) and Asp-ase (aspartic acid cleaving). One compound, 2-(Z-NH(CH2)2CONH)C6SO2F, was a potent inhibitor of Met-ase activity (k(obsd)/[I] = 162 M-1 s-1), ly-chymase activity (k(obsd)/[I] = 147 M-1 s-1), and granule-mediated as well as perforin-mediated lysis. PMSF was a poor inhibitor of granule proteases (k(obsd)/[I]'s less than 7 M-1 s-1 for four activities and no inhibition of Ser-ase); the lack of reactivity is consistent with the failure of PMSF to block granule lytic activity. We also prepared enriched perforin by anion exchange chromatography and showed that a ly-chymase and a Met-ase associated with perforin. By inhibiting these proteases we also inhibited lytic activity.
Mol Immunol 1992 Jun
PMID:Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin. 160 92

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.
J Mol Biol 1992 Jun 20
PMID:Co-operative binding of the globular domain of histone H5 to DNA. 161 93


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>