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|Title:||Studies on Acinetobacter alpha-oxoglutarate dehydrogenase.|
|Authors:||Hall, E. Rachel.|
|Presented at:||University of Leicester|
|Abstract:||A study has been made of the a-oxoglutarate dehydrogenase (a-OGDH) multi-enzyme complex from the bacterium Acinetobacter lwoffi, in order to obtain a more detailed understanding of its molecular, catalytic and, in particular, its regulatory properties. This complex, which catalyses the oxidative decarboxylation of a-oxoglutarate to produce succinyl-CoA is composed of three subunit types (E1, E2 and E3), each possessing a different enzyme activity. Comparison with the analogous complexes from E. coli and mammalian tissues has yielded both differences and similarities. The native A. lwoffi a-OGDH complex was found to have a sedimentation coefficient of 29.9S, corresponding to a molecular weight of 1.82 x 106. Electrophoresis under completely denaturing conditions revealed that the complex is composed of three discrete polypeptide chain types with molecular weights of 55000, 60080 and 80900. Using specific chemical modification it seems likely that the E3 subunit of A. lwoffi a-OGDH is similar to those of the E. coli and mammalian complexes in that they all contain a disulphide bond at the active site, which is important in the reaction mechanism. Consequently, a loss of E3 activity was observed when this S-S bond was reduced. Furthermore, by chemically modifying the E2-bound lipoic acid moiety its involvement in the reaction mechanism was also confirmed. Attempts to dissociate the complex with retention of the individual subunit activities met with only limited success. Studies into the allosteric nature of regulation of the complex yielded some indication as to the mode of action of the nucleotide effectors (NADH and AMP). Using the technique of multiple-inhibition it was shown that NADH acts at a locus other than the a-oxoglutarate binding site. In the presence of protein denaturing agents a partial desensitization to AMP was observed. A possible explanation which is consistent with these results is discussed. Finally, some doubt now exists as to whether the E1 subunit is involved in NADH regulation, since previously reported indirect kinetic evidence that NADH acts on this subunit could not be confirmed directly using a radiochemical assay for the measurement of E1 activity.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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