Biomedical subjects
Publications and source records attributed to P Dimroth.
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The biotin carboxylase component of acetyl CoA carboxylase has been purified approximately 2000 times from Escherichia coli. This protein, which catalyzes the carboxylation of free d-biotin, is free of the biotin-containing carboxyl carrier protein, is homogeneous by polyacrylamide gel electrophoresis and analytical ultracentrifugation, and has been crystallized. Biotin carboxylase, with a molecular weight of approximately 100,000, is composed of two 50,000-dalton subunits. The catalytic capacity of biotin carboxylase is markedly enhanced by ethanol (11 times at 15% v/v), and certain other organic solvents; this may mimic an effector-mediated response. The kinetic effect is exclusively on the maximal velocity of the reaction. Activation by ethanol is reversible and not accompanied by aggregation or disaggregation of the enzyme.
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The Na+-dependent citrate carrier of Klebsiella pneumoniae (CitS) is a member of the 2-hydroxycarboxylate transporter family. Within the highly conserved helix Vb region, Asn-185 of CitS was mutated to Val and Glu-194 was mutated to Gln. The wild-type and mutant proteins were synthesised in Escherichia coli DH5alpha or C43(DE3) as biotinylated or His-tagged CitS-fusions, respectively. The synthesis and purification procedure yielded 6.5 mg pure CitS per litre culture. The fusion proteins were characterised with E. coli cell suspensions or after reconstitution of the purified enzymes into proteoliposomes. The E194Q mutation had almost no effect on the kinetics of Na+ or citrate transport. In contrast, aberrant citrate transport kinetics were found for the N185V mutant. The apparent K(m) value for the citrate species H-citrate(2-) was increased about nine-fold, whereas the apparent Vmax value and the effect of Na+ on the transport kinetics were comparable to the wild-type. Asn-185 of CitS appears therefore to participate in the binding of H-citrate(2-).
In Propionigenium modestum, an Na+ cycle couples the exergonic decarboxylation of methylmalonyl-CoA to endergonic ATP synthesis. The ATPase is an F1F0-type enzyme, closely related to the F1F0 ATPase of Escherichia coli. The specificity of the P. modestum ATPase for Na+ is not absolute, as it catalyses proton transport at low Na+ concentrations. The Na(+)-binding site is located on the F0 sector. Therefore, a hybrid composed of F0 from P. modestum and F1 from E. coli, but not F1F0 from E. coli, was a functional Na+ pump. In Klebsiella pneumoniae, the Na+ ions pumped out of the cell by oxaloacetate decarboxylase are taken up again in symport with the growth substrate citrate. The reaction mechanism of oxaloacetate decarboxylase involves carboxylation of the prosthetic biotin group by carboxyltransfer from oxaloacetate, catalysed by the peripheral alpha-subunit. The firmly membrane-bound subunits beta and gamma complete the cycle by decarboxylation of the carboxybiotin intermediate which is coupled to Na+ translocation through the membrane.