Multifunctional Proteins

Kirschner, Kasper; Bisswanger, Hans

doi:10.1146/annurev.bi.45.070176.001043

Cited by 230 publications

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“…The two major advantages of oligomers seems to be cooperativity and substrate channelling. Tryptophan biosynthesis from chorismic acid in E. coli is one of the well studied pathways, where both mechanisms occur [37,38]. The extreme lability of PRA at 37°C and pH 7.5 [3] suggests that this intermediate of the tryptophan biosynthetic pathway might not normally exist in the living cell.…”

Section: Resultsmentioning

confidence: 99%

Purification and characterization of yeast anthranilate phosphoribosyltransferase

Hommel

Lustig

Kirschner

1989

European Journal of Biochemistry

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Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of M , = 83000 + 4000 (sz0,,, = 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even a< low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex formation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady-state-kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme-substrate complex, with KtNT = 1.6 pM, and KLuib-PP = 22.4 pM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylanthranilate isomerase from Escherichia coli:Much information is available on several enzymes of the tryptophan biosynthesis pathway. The structures of tryptophan synthase [l], indole-3-glycerol-phosphate (IGP) synthase and phosphoribosylanthranilate (PRA) isomerase [2] are known. Detailed kinetic studies exist on the catalytic mechanism of tryptophan synthase, IGP synthase and anthranilate synthase but not on PRA isomerase because its substrate is labile.

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Section: Resultsmentioning

confidence: 99%

Purification and characterization of yeast anthranilate phosphoribosyltransferase

Hommel

Lustig

Kirschner

1989

European Journal of Biochemistry

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“…The data suggest a dimer of a bifunctional polypeptide, with independent sites for catalysis for the two reactions. Presumably this arrangement would result in advantages to the cells in the regulation or efficiency (channeling) of these reactions (28), the consequences of which may prove to be a unique point of evolutionary divergence in the Protozoa.…”

Section: Resultsmentioning

confidence: 99%

Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata.

Ferone¹,

Roland²

1980

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

109

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“…This procedure mimics the evolution of naturally occurring bifunctional enzymes which most probably have evolved from smaller proteins through gene fusion [6]. In a recent paper 171 an approach to prepare a bifunctional P-galactosidasel galactokinase was described.…”

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Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion

Bülow¹

1987

Eur J Biochem

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An artificial bifunctional enzyme, P-galactosidaselgalactokinase, has been prepared by gene fusion. The hybrid protein catalyzes the hydrolysis of lactose followed by phosphorylation of galactose. The protein has been purified using DEAE-Sephacel chromatography and gel filtration on Sephacryl S-400 Superfine. The configuration of the hybrid protein is mainly tetrameric but also higher aggregates can be detected. The monomer M , is 160000 as judged from sodium dodecyl sulfate/polyarcylamide gel electrophoresis and the native M , has been calculated to be 600000 -650000 from gel filtration experiments. a-Galactosidase/galactokinase has different thennostability curves, pH/activity profiles and K, values as compared with the native enzymes. By using a third enzyme, galactose dehydrogenase, which competes with galactokinase for the galactose formed by /3-galactosidase, substrate channeling can be detected. This proximity effect becomes even more pronouned in an assay mixture containing poly(ethy1ene glycol).Cell metabolism is characterized by the action of enzyme sequences. From the emerging views on enzymic infrastructure it is clear that there is a spatial organization of enzymes in a metabolic sequence. The modes of such an organization entail a wide scope of protein complexes from the weak protein-protein interactions found between, for example, the glycolytic enzymes [ 11 to the multifunctional proteins possessing more than one autonomous catalytic or binding site on the same polypeptide chain.A wide variety of techniques, including immobilization of sequentially acting enzymes to a solid matrix [2, 31, crosslinking of enzymes in a random fashion [4] or in an oriented fashion with their active sites juxtaposed [5], have been applied to gain a better understanding into these proximity effects. In many of these cases, the organized enzyme systems exhibited different forms of kinetic or catalytic facilitation, compared to their counterpart systems free in bulk solution.An attractive alternative approach to the above-described systems would be to fuse two enzymes by ligating their structural genes using recombinant DNA techniques. This procedure mimics the evolution of naturally occurring bifunctional enzymes which most probably have evolved from smaller proteins through gene fusion [6]. In a recent paper 171 an approach to prepare a bifunctional P-galactosidasel galactokinase was described. The complex could catalyze the following sequential reactions :In this study an improved p-galactosidase/galactokinase hybrid protein was prepared. This artificial bifunctional enzyme was isolated from Escherichia coli carrying plasmid pZK 105 encoding an in-frame fusion between the first 1021 (out of 1023) residues of P-galactosidase followed by a 20-residue linker region and 378 (out of 382) carboxyl-terminal residues of galactokinase. In order to elucidate the microenvironmental effects caused by the gene fusion, various functional features of the /?-galactosidaselgalactokinase protein such as substrate channeling, stability...

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Multifunctional Proteins

Cited by 230 publications

References 0 publications

Purification and characterization of yeast anthranilate phosphoribosyltransferase

Purification and characterization of yeast anthranilate phosphoribosyltransferase

Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata.

Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion

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