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)

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

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