Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796809
Title: Structure-function studies of the mammalian 2-oxoglutarate dehydrogenase complex
Author: Rice, Jacqueline Ellen
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Abstract:
It has been shown that N-terminal sequence homology exists between protein X from S. cerevisiae and the bovine heart OGDC E1 enzyme. The N-terminus of OGDC E1 exhibited lipoyl-like domain characteristics; however, it was found to be non-functional as judged by its inability to be reductively acetylated in the presence of radiolabelled substrate. By degrading the bovine heart OGDC E1 enzyme specifically using trypsin, a stable lower Mr species, termed E1' was formed rapidly as a result of N -terminal cleavage of E1, generating a highly- immunogenic peptide with an approx. Mr value of 8,000. Tryptic degradation of OGDC also resulted in the removal of the lipoyl domain from E2 at longer time intervals, the E3 enzyme remaining intact under these conditions. On gel permeation chromatography of trypsin-treated complex where E1' had been formed, the E3 enzyme was found to exhibit a reduced affinity for the complex; in addition E1' also showed a marked tendency to dissociate from the E2 core assembly. Similarly, protease arg C treatment of OGDC causing extensive but specific cleavage of E1 only, also resulted in release of E3 from the core. On treatment with trypsin, the overall activity of OGDC was found to decrease. This decline in activity was not caused by inactivation of E1 as the modified E1 enzyme retained approx. 100% activity. Loss of OGDC function is instead attributed to the reduced affinity of the E3 (and E1) enzymes for the core structure. There are indications that E1'/E1 heterodimers of E1 may be able to sustain overall complex activity. From the above experiments coupled to sequence alignment analysis of protein X from S. cerevisiae, it is proposed that a truncated, non-functional 'lipoyl-like' domain is situated at the N-terminus of the E1 enzyme of OGDC. Evidence is presented that this region is followed closely by an E3 binding domain, cleavage of which, results in E3 and E1 dissociation from the complex. The exact mode of association which exists between E1 and E3 within OGDC has not, as yet, been clearly defined. Dissociation of intact OGDC in conditions of high ionic strength has no significant effect on complex activity; however, after tryptic degradation of OGDC to varying extents to form differing amounts of the Elo species, the residual complex activity exhibits an increased salt sensitivity profile. It was also shown that treatment of the complex with 0.25M MgCl2 representing high ionic strength, was sufficient to render the E3 enzyme susceptible to inhibition by N- ethylmaleimide but only in an NADH dependent manner. Similar partial protection of the redox-active disulphide cysteine pair on E3 in the intact complex was observed in the presence of the specific active-site inhibitor p- aminophenyldichloroarsine. These findings are consistent with the view that the active-site of E3 is shielded from the external environment in the intact complex. In bovine heart PDC, it has been proposed that protein X performs a role in protecting the active site of E3. Since it is postulated that protein X in this complex and the N-terminal region of bovine heart OGDC E1 are both involved in binding E3, the possible role of the N-terminal region of OGDC E1 in the active site protection of E3 was investigated. The E3 enzyme of OGDC was found to be susceptible to inhibition by N-ethylmaleimide in an NADH dependent manner when the complex was treated with high salt concentrations; in contrast, protease treatment of the complex with trypsin had little effect on E3 activity in the presence of NADH and N-ethylmaleimide. Protease arg C treatment, however, allowed N-ethylmaleimide in the presence of NADH to effect an overall reduction in complex activity of approx. 50%. These results indicate that the large E1' species may still protect released E3 from modification by N-ethylmaleimide. However, no evidence was obtained for the presence of a stable ElV E3 subcomplex. The importance of the N-terminal region of E1 of OGDC in mediating the re-folding of the complex after denaturation of the enzyme with GdnHCl or in regulating its sensitivity to was investigated. It was established that this region was not critical to the stability of the enzyme or its capacity to renature. The Ca2+ dependence of its activity also appeared to be unaltered. Such data are consistent with the view that the primary role of this region of the E1 enzyme is a structural one in promoting E3 and E1 interactions with the core of the complex.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796809  DOI: Not available
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