Studies on a Mutant Form of Escherichia coli Citrate Synthase Desensitised to Allosteric Effectors

Danson, Michael J.; Harford, Stephen; Weitzman, P.D.J.

doi:10.1111/j.1432-1033.1979.tb19746.x

Cited by 25 publications

(14 citation statements)

References 20 publications

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“…Studies with the purified E. coli enzyme have revealed a number of potential effectors that may be involved in this regulation. These include 2-ketoglutarate, NADH, and ATP (28,29). When glucose plus acetate are the carbon sources, the velocity through the Krebs cycle is substantially lower and the cycle has a secondary role in energy production and biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

Characterization of rate-controlling steps in vivo by use of an adjustable expression vector.

Walsh¹,

Koshland²

1985

Proc. Natl. Acad. Sci. U.S.A.

126

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Citrate synthase (EC 4.1.3.7) was varied from 10% to 5000% the level found in wild-type Escherichia coli by means of recombinant DNA techniques. When acetate was the sole carbon source, cell growth and carbon flow through the Krebs cycle were greatly affected by the underproduction of citrate synthase. In contrast, when glucose was the main nutrient, the same underproduction of citrate synthase had little effect on either growth or carbon flux. When the enzyme was overproduced 50-fold, cultures would grow on glucose but cell division could be abruptly stopped by adding acetate to the medium. These results indicate that the regulatory properties of citrate synthase are highly dependent on the carbon-source composition of the medium. Furthermore, recombinant DNA technology can be used to alter rate-controlling steps in biological pathways and elucidate the regulatory properties of metabolic systems.Development, growth, and maintenance metabolism all involve differential expression of biological pathways. Pathways are in turn controlled by enzymatic steps whose individual properties determine the overall rate of the process. In cancer and inborn metabolic diseases, aberrations in one or more steps of certain pathways cause abnormal or deficient growth. In some cases this seems to be caused by overexpression or underexpression of a normal enzyme (1, 2). To understand the underlying causes of these pathological conditions, it is necessary to identify which enzymes control the relevant metabolic processes and to determine how alterations in their levels affect the network of interactions inside the cell.The concept of "rate-controlling" steps has been widely applied to describe the potential control points in metabolic pathways, protein secretion, or macromolecule biosynthesis (3). Generally, a rate-controlling enzyme is assumed to catalyze the "slow" step in a pathway and a modulation of its properties will control the overall rate of the system. These enzymes tend to be points of regulation because the rate of the process will be most sensitive to control at these steps. Enzymes classified as non-rate-controlling are present at a catalytic excess so that change in the kinetic properties of such enzymes would have a minimal impact on the overall velocity of the system. Kacser and Burns (4) and Heinrich and Rapoport (5, 6) have pointed out that rarely is a single enzyme rate-controlling in a pathway. They have developed a theoretical analysis of pathway fluxes and defined a sensitivity coefficient that expresses the degree to which individual steps influence the overall rate through the pathway. The sum of the sensitivity coefficients of all the enzymes in a pathway will be equal to 1.

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Section: Discussionmentioning

confidence: 99%

Characterization of rate-controlling steps in vivo by use of an adjustable expression vector.

Walsh¹,

Koshland²

1985

Proc. Natl. Acad. Sci. U.S.A.

126

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“…That is, it is possible to generate a citratesynthase-negative srrain of E. coli by mutation within the gltA gene, as 2-methylcitrate synthase (which also exhibits citrate synthase activity) is not produced unless propionate is present in the growth medium. Furthermore, when we mutated the citrate-synthase-negative strain to create a revertant that had regained the citrate synthase activity (Danson et al, 1979), we presumably had caused the constitutive expression of the 2-methylcitrate synthase gene. Consequently, this revertant still produced an inactive hexameric citrate synthase, the gene sequence of which was identical to that of the original citrate-synthase-negative parent (Patton et al, 1993), plus the 2-rnethylcitrate synthase with its associated citrate synthase activity.…”

Section: Ds2-3r Grown On Nutrient Brothmentioning

confidence: 99%

Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships

et al. 1998

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Following the complete sequencing of the Escherichia coli genome, it has been shown that the proposed second citrate synthase of this organism, recently described by the authors, is in fact a 2-methylcitrate synthase that possesses citrate synthase activity as a minor component. Whereas the hexameric citrate synthase is constitutively produced, the 2-methylcitrate synthase is induced during growth on propionate, and the catabolism of propionate to succinate and pyruvate via 2-methylcitrate is proposed. The citrate synthases of the psychrotolerant eubacterium DS2-3R, and of the thermophilic archaea Thermoplasma acidophilum and pyrOcoccus furiosus, are approximately 40 O/ o identical in sequence to the Escherichia coli 2-methylcitrate synthase and also possess 2-methylcitrate synthase activity. The data are discussed with respect to the structure, function and evolution of citrate synthase and 2-methylcitrate synthase.

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“…The small enzyme is senlsitive in vitro to inhibition by ATP but not by at-ketoglutarate or NADH, while the large enzyme is sensitive in vitro to inhibition by a-ketoglutarate and NADH but not by ATP. In addition, the two types differ in their binding affinity for the substrate acetyl coenzyme A (acetyl-CoA) (2,7,24). Interestingly, the mnonomers of both the large and small enzymes are comparable in size and share amino acid sequence homology (3,4,22,41).…”

mentioning

confidence: 99%

Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene

Wood¹,

Williamson²,

Winkler³

et al. 1987

J Bacteriol

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The Rickettsia prowazekii citrate synthase (gitA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomnal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pafrs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli Snd pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyine residUes. Upstream from the open reading frame and in close proximity to one anothet, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demobstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial git4 gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.Rickettsia prowazekii is an obligate intracellular parasitic bacterium that multiplies within the cytoplasm of its eucaryotic host cell rather than within a phagosome or phagolysosome. Its exploitation of this environment is reflected in a variety of novel features, most notably the existence of an ADP-ATP transport system that permits the rickettsiae to accumulate ATP directly from the cytoplasm of the host cell (44). However, the rickettsiae are not strict energy parasites and can generate their own ATP via the tricarboxylic acid cycle and oxidative phosphorylation (39). This ability is probably necessary for cell viability during periods when the host cell ATP reserves are depleted or during transit of the bacterium to a new host cell. Central to the regulation of the tricarboxylic acid cycle is the enzyme citrate synthase.Citrate synthase, the first enzyme of the tricarboxylic acid cycle, has been extensively studied in a variety of procaryotic and. eucaryotic cells (30,, 41, 43). These studies have identified two main types of citrate synthase: a "small" type (molecular weight, -106,000) found in eucaryotic cells and gram-positive bacteria and a "large" type (molecular weight, -250,000) found exclusively in gram-negative bacteria. The small enzyme is a dimer composed of two identical subunits, while the large enzyme is a multimer composed of four to six identical subunits. The two types of enzyme also differ in their sensitivity to certain effectors. The small enzyme is senlsitive in vitro to inhibition by ATP but not by at-ketoglutarate or NADH, while the large enzyme is sensitive in vitro to inhibition by a-ketoglutarate and NADH but not by ...

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Studies on a Mutant Form of Escherichia coli Citrate Synthase Desensitised to Allosteric Effectors

Cited by 25 publications

References 20 publications

Characterization of rate-controlling steps in vivo by use of an adjustable expression vector.

Characterization of rate-controlling steps in vivo by use of an adjustable expression vector.

Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships

Nucleotide sequence of the Rickettsia prowazekii citrate synthase gene

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