The quaternary structures of pyruvate dehydrogenase complex (PDH complex) and 2-ketoglutarate dehydrogenas complex (KDH complex) from E. coli will be further anlayzed by electron microscopy. The structural model recently developed for the PDH complex using scanning transmission electron microscopy (STEM) and radial mass analysis will be further tested. Similar methods will be applied to quaternary structural analysis of the KDH complex in which the arrangement of 2-ketoglutarate dehydrogenase (E1) subunits about the cubic core protein dihydolipoly transsuccinylas (E2) should differ from that observed for the corresponding proteins in the PDH complex. The locations of the lipoyl groups attached to the dihydrolipoyl transacetylas (E2) component of PDH complex relative to pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) subunits will be established by labeling the dihydrolipoyl moieties with undecagold cluster complexes and carrying out radial mass analysis using STEM. Undecagold cluster complexes prepared as monofunctional acylating and alkylating agents will be used to label lipoamide moieties in the PDH complex. The positions of these labels relative to E1 and E3 will be mapped by radial mass analysis using STEM. The mechanism of reductive acylation between E1 and E2 of the PDH complex will be investigated, with reference to the question of the mechanistic relationship between electron transfer and group transfer. To this end, 2-acetyl-TPP will be prepared and its enzymatic and nonenzymatic properties investigated. Trapping experiments with [2-14C]pyruvate and PDH complex will be carried out in search of [1-14C]acetyl-TTP in the steady state. A radical cation form of 2(1-hydroxyethylidene)-TTY will be sought in experiments with E1, ferricyanide and pyruvate. Spin trapping experiments will also be carried out. Suicide inactivation of the PDH complex by 3-fluoropyruvate and by pyruvate plus 02 will be characterized to determine the inactivation mechanisms. Lysin 2,3-aminomutase will be purified from Clostridia and further characterized, with special reference to its metal ion content and the possible presence of a Fe-S cluster. The reaction mechanism will be investigated by determining the tritium-kinetic isotope effect for the reaction of beta-[2-3H]lysine. The involvement of pridoxal phospate in this B12-like reaction will also be investigated.
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