Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.
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PMID:Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex. 150 15

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.
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PMID:Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis. 318 51

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.
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PMID:Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. 342 11

A computer modeling system developed to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) accompanying release of lipoyl moieties by lipoamidase and by trypsin [Hackert, M.L., Oliver, R.M. & Reed, L.J. (1983) Proc. Natl. Acad. Sci. USA 80, 2226-2230] was used to analyze analogous data for the E. coli pyruvate dehydrogenase complex (PDC). The model studies indicate that the activity of PDC, as found for KGDC, is influenced by redundancies and random processes, which we describe as a multiple random coupling mechanism. In both complexes more than one lipoyl moiety services each pyruvate dehydrogenase (EC 1.2.4.1) or alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) (E1) subunit, and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data for PDC was obtained with a model that has four lipoyl domains with four or, more probably, eight lipoyl moieties servicing each E1 subunit. The lipoyl-lipoyl interaction network for PDC has lipoyl domain interactions similar to those found for KGDC plus the additional possibility of interaction of a lipoyl moiety and its paired mate on each dihydrolipoamide acetyltransferase (EC 2.3.1.12) (E2) subunit. The two lipoyl moieties on an E2 subunit in PDC appear to be functionally indistinguishable, each servicing the acetyltransferase site of that E2 subunit and a dihydrolipoamide dehydrogenase (EC 1.6.4.3) (E3) subunit if the latter is bound to that particular E2 subunit. The observed difference between inactivation of PDC by lipoamidase and by trypsin appears to be due to dead-end competitive inhibition by lipoyl domains that have been modified by excision of lipoyl moieties by lipoamidase.
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PMID:A computer model analysis of the active-site coupling mechanism in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. 634 73

The alpha-ketoglutarate dehydrogenase complex from Escherichia coli consists of a core component, dihydrolipoyl transsuccinylase (E2), to which are noncovalently bound 12 polypeptide chains each of alpha-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase. E2 exists as a cube-shaped complex comprising 24 identical chains and may be resolved from the other two enzyme components. Limited digestion of E2 with trypsin quantitatively removes domains containing the lipoic acid cofactor while leaving the quaternary structure of the complex intact. Averages of native and trypsin-modified E2 were computed from images of single molecules obtained from electron micrographs of negatively stained specimens. The two averages were very similar and were in general agreement with a model determined previously by X-ray crystallography. However, detailed analysis of the difference image, obtained by subtracting the average of the trypsin-treated E2 from the native E2, showed extra stain-excluding regions along the edges of the native molecule which we interpret as representing the lipoyl-bearing domains. Micrographs of mixtures of native and modified E2 were also analyzed in order to rule out staining or electron-optical artifacts as accounting for the results. On the basis of these results along with other available structural information, we propose that one function of the lipoyl domains is to permit interactions between distantly separated lipoyl moieties in the E2 complex; this proposal also agrees with recent results of modeling studies of biochemical data [Hackert, M.L., Oliver, R.M., & Reed, L.J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2226-2230].
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PMID:Localization of lipoyl-bearing domains in the alpha-ketoglutarate dehydrogenase multienzyme complex. 638 May 87

A computer modeling system was used to analyze experimental data for inactivation of the Escherichia coli alpha-ketoglutarate dehydrogenase complex accompanying release of lipoic acid residues by lipoamidase and by trypsin [Stepp, L. R., Bleile, D. M., McRorie, D. K., Pettit, F. H. & Reed, L. J. (1981) Biochemistry 20, 4555-4560]. The results provide insight into the active-site coupling mechanism in the alpha-ketoglutarate dehydrogenase complex. The model studies indicate that the overall activity of the alpha-ketoglutarate dehydrogenase complex is influenced by redundancies and random processes that we describe as a multiple random coupling mechanism. More than one lipoyl moiety services each E1 subunit (alpha-ketoglutarate dehydrogenase, EC 1.2.4.2), and an extensive lipoyl-lipoyl interaction network for exchange of electrons and possibly acyl groups must also be present. The best fit between computed and experimental data was obtained with a model that has two lipoyl moieties servicing each E1 subunit and a lipoyl-lipoyl interaction network that links all lipoyl moieties on the E2 cube (dihydrolipoamide succinyltransferase, EC 2.3.1.61). The single lipoyl moiety on an E2 subunit is assumed to service the coenzyme A-dependent succinyltransferase site of that E2 subunit as well as an E3 subunit (dihydrolipoamide dehydrogenase, EC 1.6.4.3) if the latter is bound to that particular E2 subunit.
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PMID:Evidence for a multiple random coupling mechanism in the alpha-ketoglutarate dehydrogenase multienzyme complex of Escherichia coli: a computer model analysis. 640 46

Branched-chain alpha-ketoacid dehydrogenase has been purified to homogeneity from bovine liver mitochondria. The isolated complex has a specific activity of 5-8 mumol of reduced nicotinamide adenine dinucleotide min-1 (mg of protein)-1 as isolated and does not require the addition of exogenous lipoamide dehydrogenase for activity. Addition of porcine heart lipoamide dehydrogenase stimulated complex activity by no more than 20%. Four subunits copurify with the complex with molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 55 000, 52 000, 46 500, and 37 500. Here we show that the 52 000-dalton subunit is the lipoyl-containing transacylase component of the complex. Data are presented to support the hypothesis that the branched-chain ketoacid dehydrogenase complex is physically and catalytically similar to, but separate from, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The transacylase of the branched-chain ketoacid dehydrogenase complex has an exposed trypsin-sensitive region. Proteolytic action of trypsin separates a lipoyl-containing component from the remainder of the protein. Data from our laboratory presented here and elsewhere define a specific function for three of the four subunits.
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PMID:Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase. 665 74

The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-[[[(iodoacetyl)amino]ethyl]amino]-naphthylene-1-sulfonic acid. Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns. This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex. The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1. In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe. At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds. The increase in the correlation time probably reflects interactions between pyrene moieties. The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns. This suggests that the native structure is necessary for observation of lipoic acid movement within the complex. Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments. The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.
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PMID:Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli. 675 46

The relationships between release of (3)H-labeled lipoyl moieties by trypsin and lipoamidase and accompanying loss of overall enzymatic activity of the Escherichia coli pyruvate and alpha-ketoglutarate dehydrogenase complexes were studied. Trypsin releases lipoyl domains together with their covalently attached lipoyl moieties from the "inner" core of the dihydrolipoyl transacetylase and the dihydrolipoyl transsuccinylase whereas lipoamidase releases only the lipoyl moieties. The results show that release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at faster rates than the accompanying loss of overall activity of the two complexes. Trypsin released about half of the lipoyl domains in the pyruvate dehydrogenase complex without significant effect on the overall activity. A model is presented to explain these and other observations on active-site coupling via lipoyl moieties.
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PMID:Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli. 679 98

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.
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PMID:Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli. 680 71


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