U419836.pdf (311.11 MB)
Studies on Escherichia coli citrate synthase.
thesisposted on 2015-11-19, 09:08 authored by Michael John. Danson
I Citrate synthase from Escherichia coli has been purified to homogeneity. Gel electrophoresis indicated that the enzyme exists in two or three electrophoretically separable forms in equilibrium with each other. Ultracentrifugation studies showed a size difference between these species: monomer (mol.wt. = 57,000), tetramer (mol.wt. = 230,000) and possibly a higher molecular weight protein. The tetramer is the predominant species, accounting for over 75 of the total protein at pH 8.0, and active-enzyme centrifugation demonstrated that it possesses catalytic activity. Electrophoresis in the presence of sodium dodecyl sulphate suggested that the subunits are of similar size with a molecular weight of approximately 55,000. Examination of the enzyme by electron microscopy after shadowing with platinum confirmed the predominance of the tetramer and further suggested that the subunits may be arranged in a square.;II Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and ?-oxoglutarate. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependences of their rates of photo- -oxidation with Methylene Blue suggest that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal. In addition, photo-oxidation suggests the participation of histidine at the ?-oxoglutarate effector site and cysteine at the NADH site. Amino acid analyses of native and photo-oxidized enzymes were consistent with these conclusions. Treatment of citrate synthase with diethyl pyrocarbonate and subsequent analysis of the modified enzyme confirmed the involvement of histidine in catalytic activity. Modification with 2-hydroxy-5-nitrobenzyl bromide also indicated the participation of tryptophan in the activity of the enzyme. Studies with the thiol-specific reagent DTNB were consistent with the photo-oxidation experiments in suggesting that cysteine residues are essential for NADH inhibition. Furthermore, the inactivation of the enzyme by DTNB indicated the additional involvement of cysteine in catalytic activity. A detailed kinetic analysis of the modification of the enzyme by DTNB is presented. The possible roles of these functional residues in the activity and regulation of citrate synthase are discussed.;III The effects of KCl on citrate synthase have been investigated. Examination of the enzyme by gel electrophoresis and gel filtration suggested that in the presence of KCl only the tetrameric species is present. Moreover, ultracentrifugation studies have demonstrated that the salt also induces the tetramer to assume a more compact conformation. Further evidence for these salt-induced changes has been gained from studies on the reactivity of cysteine residues to modification by DTNB and from an investigation of the exposure of tyrosine and tryptophan residues by solvent perturbation difference spectroscopy. Finally, preliminary kinetic studies indicated that the activation of citrate synthase by KCl is the result of an approximately 5-fold reduction in the Km for acetyl-CoA. The possible ways in which KCl and other neutral salts affect the structure of citrate synthase are discussed.;IV Preliminary investigations of the citrate synthases from two other Enterobacteriaceae (Serratia marcescens and Klebsiella aerogenes) have been made. The two enzymes have been purified and each shown to exist in an equilibrium between three electrophoretic species. Modification of these enzymes with DTNB has shown that, as with the citrate synthase of E.coli, thiols are required for full enzymic activity and sensitivity to NADH.
Date of award1974-01-01
Awarding institutionUniversity of Leicester