The enzymes of the glyoxylate bypass operon of Escherichia coli ML308

MacKintosh, Carol (1987) The enzymes of the glyoxylate bypass operon of Escherichia coli ML308. PhD thesis, University of Glasgow.

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1. The glyoxylate bypass allows Escherichia coli to generate precursors for biosynthesis during growth on acetate. During operation of the bypass there is competition for isocitrate between isocitrate dehydrogenase (ICDH) and isocitrate lyase (ICL). Previous studies had shown that ICDH is regulated by a phosphorylation/dephosphorylation mechanism. However, ICL and its possible contribution to regulation at this branchpoint had not been studied. 2. A quick, reliable procedure was developed for purification of ICL from acetate-grown Escherichia coli ML308. 10-12 mg of pure enzyme could be obtained from 20g wet weight of bacteria. The specific activity was 35-40mumol/min/mg of protein at 37 C. 3. The of the native enzyme was 177 000, determined by gel filtration and 180 000, determined by analytical ultracentrifugation. The subunit Mr. was 45 000. This implies that Escherichia coli ICL is a tetramer. 4. The optimum pH for ICL activity was pH 7.3, within the physiological range for Escherichia coli. 5. The kinetics of binding of substrates, products and/or their analogues was studied for the cleavage and the condensation reactions of ICL. The results show that, unlike the enzyme from other sources, ICL from Escherichia coli obeys a random order mechanism in which an enzyme-isocitrate-succinate ternary complex can be formed. 6. The Km of ICL for Ds -isocitrate was 0.063mM at pH7. 3. The Km was sensitive to changes in pH and also to the presence of inorganic anions such as Cl- and SO2-. 7. Several compounds were found to inhibit ICL but these effects could be ascribed to structural similarities between the inhibitors and the substrates for the enzyme. 2-oxoglutarate and phosphoenolpyruvate were shown to be succinate analogues and 3-phosphoglycerate was shown to be a glyoxylate analogue. The intracellular concentration of 3-phosphoglycerate suggests that this compound may inhibit ICL significantly in intact cells. However, none of the effects observed could be attributed to the existence of an allosteric regulatory site. 8. There was no evidence that ICL is a phosphorylated molecule nor that ICDH kinase/phosphatase can affect ICL. 9. A recombinant plasmid carrying an 11 kilobase Clal-Clal fragment of genomic DNA which complements an aceA (ICL) mutation was constructed by Dr. E. M. T. El-Mansi. Measurement of enzyme activities in crude cell extracts and in vitro transcription-translation experiments showed that this plasmid (pEM9) encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (MS-A), ICL and ICDH kinase/phosphatase and in that order. 10 ICL was purified from the overexpressing strain KAT-1/pEM9 following the procedure developed for ICL from Escherichia coli ML308 up to the phenyl-Sepharose step. ICL from both sources was identical by peptide-mapping, Km, NH2-terminal sequence and amino acid composition. The pI of both ICLs was 4. 4 as determined by chromatofocusing on a Fast Protein Liquid Chromatography Mono P column. 11. Overexpression of MS-A in Escherichia coli KAT-1/pEM9 meant that it could be distinguished from MS-B and a purification was developed for MS-A from Escherichia coli KAT-l/pEM9. The N-terminal amino acid sequence of MS-A was determined. 12. ICDH kinase/phosphatase from Escherichia coli KAT-1/pEM9 was subjected to limited proteolysis by trypsin. Proteolysis proceeded in two stages in which the first cleavage product had lost its ICDH phosphatase activity but retained its ICDH kinase activity. The second cleavage product was completely devoid of both kinase and phosphatase activities. These results could mean that the active sites for ICDH kinase and ICDH phosphatase are different.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76647
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:58
Last Modified: 19 Nov 2019 13:58

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