A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase

Hankins, Charles N.; Largen, Michael; Mills, Stanley E.

doi:10.1016/0003-2697(75)90154-2

Cited by 18 publications

(14 citation statements)

References 6 publications

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“…The reaction catalyzed by IGPS ( Fig. 1) can be followed fluorimetrically by exciting the fluorescence of the product IGP (Hankins et al, 1975). Moreover, the substrate analogue rCdRP (Bisswanger et al, 1979) is a useful fluorescent ligand to probe the active site of eIGPS by binding studies Eberhard et al, 1995).…”

Section: Enzyme Kinetic Constantsmentioning

confidence: 99%

“…During purification, protein concentrations were estimated using the BioRad protein assay (Bradford, 1976) with BSA as standard. Enzymatic activity of IGPS variants using enzymatically produced CdRP was monitored fluorimetrically (Hankins et al, 1975) as described by Hommel et al (1995) and Eberhard et al (1995). The steady state kinetic constants were determined from initial velocities or by evaluation of entire progress curves (Duggleby & Morrison, 1977).…”

Section: Production and Purification Of Eigps Variantsmentioning

confidence: 99%

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Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

et al. 1998

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Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-l-deoxyribulose 5 ' -phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state ktnetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.

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Section: Enzyme Kinetic Constantsmentioning

confidence: 99%

Section: Production and Purification Of Eigps Variantsmentioning

confidence: 99%

Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

et al. 1998

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“…PRA isomerase (16 pg) was measured in an identical protocol. fold-higher value than that determined for the Escherichia coli enzyme (7,10). Thus, the high CDRP substrate concentration in routine assays precluded the use of a spectrophotofluorimetric assay for the B. subtilis InGP synthase (10) because the product InGP fluorescence is strongly quenched at the CDRP concentrations.…”

Section: Resultsmentioning

confidence: 95%

“…fold-higher value than that determined for the Escherichia coli enzyme (7,10). Thus, the high CDRP substrate concentration in routine assays precluded the use of a spectrophotofluorimetric assay for the B. subtilis InGP synthase (10) because the product InGP fluorescence is strongly quenched at the CDRP concentrations. Optimal assay conditions for the B. subtilis PRA isomerase were essentially the same as described for the Pseudomonas putida enzyme (5).…”

Section: Resultsmentioning

confidence: 95%

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis

Hoch¹

1979

J Bacteriol

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Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The

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“…PRAI activity was determined spectrophotometrically at 278 nm using a 0.1 mg/ml concentration of a PRAI-deficient E. coli helper cell extract (W3110 trp C 9830 (F Ϫ )) (11). To determine IGPS activity both a spectroscopic (13) and a fluorimetric assay system (14) were used.…”

Section: Methodsmentioning

confidence: 99%

Multifunctional Tryptophan-synthesizing Enzyme

Schwarz

Uthoff

Meyer

et al. 1997

Journal of Biological Chemistry

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After developing a suitable procedure to produce large amounts of Euglena gracilis as well as a reliable protocol to purify the multifunctional tryptophan-synthesizing enzyme derived from it (Schwarz, T., Bartholmes, P., and Kaufmann, M. (1995) Biotechnol. Appl. Biochem. 22, 179 -190), we here describe structural and catalytic properties of the multifunctional tryptophansynthesizing enzyme. The kinetic parameters k cat of all five activities and K m for the main substrates were determined. The relative molecular weight under denaturing conditions as judged by SDS-polyacrylamide gel electrophoresis is 136,000. Cross-linking as well as gel filtration experiments revealed that the enzyme exists as a homodimer. Neither intersubunit disulfide linkages nor glycosylations were detected. On the other hand, the polypeptide chains are blocked N-terminally. Complete tryptic digestion of the protomer, high pressure liquid chromatography separation of the resulting peptides, and N-terminal sequence analysis of homogenous peaks as judged by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry was performed. Depending on the sequenced peptides, alignments to all entries of the SwissProt data base resulted in both strong sequence homologies to known Trp sequences and no similarities at all. Proteolytic digestion under native conditions using endoproteinase Glu-C uncovered one major cleavage site yielding a semistable, Nterminally blocked fragment with a molecular weight of 119,000. In addition, an increase in ␤-elimination accompanied by a decrease in ␤-replacement activity of the ␤-reaction during proteolysis was observed. In most procaryotes, tryptophan synthase is organized as a tetrameric bienzyme complex consisting of a central ␤ 2 -dimer (TRPS␤) combined with two peripheral ␣-subunits (TRPS␣). The Escherichia coli and Salmonella typhimurium enzymes in particular have been intensively investigated in the past (1, 2). In fungi such as Saccharomyces cerevisiae and Neurospora crassa, tryptophan synthase is a dimer of identical bifunctional polypeptides (3,4,5). In contrast, the enzyme isolated from Euglena gracilis, here designated as multifunctional tryptophan-synthesizing enzyme (MTSE), is capable of catalyzing all reactions shown in Scheme I. Although this discovery was made 2 decades ago (6, 7), no thorough further characterization of this multifunctional enzyme has been published so far, probably due to the excessive expenditure to cultivate sufficient amounts of E. gracilis and the difficulties of purifying MTSE with outdated technology. Using state of the art equipment and materials, we established a reliable and simple procedure to obtain MTSE from E. gracilis (8). Here we present both a reinvestigation of earlier initial findings and new data with respect to structural and functional properties of the enzyme. EXPERIMENTAL PROCEDURESChemicals, Enzymes, and E. coli Strains-If not otherwise stated, all chemicals (reagent or ultrapure grade) were obtained from Sigma (Deisenhofen, Germany) or Merck...

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A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase

Cited by 18 publications

References 6 publications

Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis

Multifunctional Tryptophan-synthesizing Enzyme

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