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Title: Regulation of the isocitrate and [alpha]-ketoglutarate dehydrogenases from Acinetobacter lwoffi.
Authors: Parker, George Malcolm.
Award date: 1972
Presented at: University of Leicester
Abstract: 1. Isoenzymes of NADP-linked isocitrate dehydrogenase, termed IDH-I and IDH-II have been shown to exist in Acinetobacter lwoffi (Self, 1969). The isoenzymes were separated using zonal ultracentrifugation and IDH-II was found to be stimulated by glyoxylate or pyruvate. In the present work, an improved separation procedure has been devised and the regulatory properties of the isoenzymes further investigated. Low concentrations of AMP and ADP stimulate IDH-II by an increase in Vmax but have no effect on IDH-I. Other changes brought about by AMP and ADP include a shift in pH optimum for enzyme activity and an increase in the stability to thermal inactivation. The activation of the enzyme by AMP/ADP and pyruvate/glyoxylate has been shown to be quite independent and the allosteric nature of the regulatory sites has been demonstrated by differential desensitisation of the enzyme with urea. The physiological significance of the activations is discussed. 2. The ?-ketoglutarate dehydrogenase complex from A.lwoffi has been purified to homogeneity as indicated by gel electrophoresis and sedimentation velocity analysis. The enzyme has a S20,w value of 29s and is a polyhedron of 300? diameter. The Km values for ?-ketoglutarate, NAD and CoASH were 2.0mM, 0.10mM and 0.012mM respectively. Preliminary observations which had been carried out with unpurifiod preparations (Weitzman, 1972) were confirmed and indicated the following regulatory properties:- (a) AMP and ADP stimulate enzyme activity by a reduction of the apparent Km for ?-ketoglutarate, while ATP is slightly inhibitory; (b) NADH inhibits the enzyme; (c) AMP and ADP overcome the NADH inhibition. The sites of action of the effectors have been investigated by means of assays specific for the component enzymes and by kinetic studies. El (?-ketoglutarate decarboxylase) was assayed by monitoring the oxidative decarboxylation of ?-ketoglutarate with dichlorophenolindophenol. A new assay procedure has been devised for E3 (lipoamide dehydrogenase) which monitors the appearance of oxidised lipoamide polarographically. The validity of the assay has been demonstrated and the principles and advantages of it are discussed. The site of action of NADH was further investigated by performing product inhibition studies with NADH on the whole enzyme complex. Both El and E3 were inhibited by NADH whereas only El was stimulated by AMP and ADP, E3 being unaffected by these nucleotides. The allosteric nature of the adenylate regulation has been shown by the desensitisation of the enzyme with urea. This desensitisation is accompanied by a reduction of the apparent Km for ?-ketoglutarate. The physiological significance of the regulation is discussed. 3. A large number of bacterial genera has been examined to investigate the variations in the regulatory behaviour of the isocitrate and ?-ketoglutarate dehydrogenases. The regulatory properties of pyruvate dehydrogenase, citrate synthase and succinic thiokinase are also tabulated and comparisons made. Most striking was the observation that all five enzymes in the genus Acinetobacter and a number of other bacteria were regulated by adenylates in a similar manner to that observed in A.lwoffi. .
Level: Doctoral
Qualification: Ph.D.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biology
Leicester Theses

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