Biomedical subjects
Publications and source records attributed to B E Davidson.
The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.
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The following amino acid sequence information has been determined for the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus cremoris US3: the C-terminal amino acid, the N-terminal sequence of the first 20 amino acids and the sequence of a 53-residue tryptic peptide containing the only cysteine residue in the protein. The enzyme was cleaved by alkali at the cysteine residue following reaction first with 5,5'-dithiobis(2-nitrobenzoic acid) and then with K14CN. This treatment yielded two cleavage products as well as some higher polymers and some uncleaved enzyme. The radioactive cleavage product was purified and its size indicated that the cysteine residue is 80 residues from the C-terminus. Comparisons of the sequences determined for the S. cremoris enzyme with those already known for dogfish lactate dehydrogenase indicate that the two enzymes are only distantly related since the sequence homology between them is limited and of borderline statistical significance.
The amino acid sequences of 28 soluble tryptic peptides from chorismate mutase/prephenate dehydratase from Escherichia coli K12 have been determined. Together with the four unique cysteine-containing peptides sequenced by Gething and Davidson ((1976) Eur. J. Biochem. 71, 327-336) this accounts for approximately 75% of the total sequence expected for this protein. A high frequency of identify between some of the peptides suggests the possibility of gene duplication during the evolution of the structural gene for the enzyme.
The effects of phenylalanine, NaCl and pH on the conformation of chorismate mutase/prephenate dehydratase have been investigated, using measurements of far and near-ultraviolet circular dichroic spectra and ultraviolet difference spectra. At pH 8.2 in 20 mM Tris-Cl buffer the enzyme was found to contain 10-20% helix and 40-50% beta-structure. There was little or no change in these values on the addition of 1 mM phenylalanine (the allosteric effector) or 0.4 M NaCl or by decreasing the pH to 7.4. Both phenylalanine and NaCl caused significant changes in the conformation of the enzyme. The most prominent of these was the movement of a tryptophan residue into a more hydrophobic environment. There was also a slight perturbation of this tryptophan when the pH was decreased to 7.4. The conformational changes can explain sigmoidal kinetic behaviour observed previously [Gething et al. (1976) Eur. J. Biochem. 71, 317-325].
The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady-state dialysis. Under most of the experimental conditions examined positive co-operativity was observed for the binding of ligand up to 50% saturation and negative co-operativity above 50% saturation. In the presence of 0.4 M NaCl at pH 8.2 the co-operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine-binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities. The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5'-dithio-bis (2-nitrobenzoic acid) did not bind phenylalanine. The mechanism of co-operativity, the binding of the enzyme to Sepharosyl-phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.
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Pyrrolidonecarboxylate peptidase (EC 3.4.11.8) from Streptococcus faecium was purified by fractionation with streptomycin sulphate and ammonium sulphate, by chromatography on Sephadex G200 and DEAE-cellulose, and by preparative electrophoresis on Sephadex G25. The purified enzyme on acrylamide gel showed a strong protein band which contained enzyme activity and a very faint band which had no activity. The subunit molecular weight of the purified enzyme was estimated by acrylamide gel electrophoresis in sodium dodecyl sulphate to be 42,000 +/- 1,000. The enzyme showed optimum activity at pH 7.6 and was unstable in the absence of 2-mercaptoethanol. The sensitivity of the enzyme to alkylating agents (N-ethylmaleimide and iodoacetamide) suggested that free sulphydryl groups were essential for enzyme activity. The enzyme was rapidly inactivated above 45 degrees C. The values of the Michaelis constants (Km) obtained with various L-pyrrolidonecarboxylyl dipeptides were similar although there was a 10-fold range in the maximal rates of hydrolysis of these substrates. Inhibition studies showed that the substrate analogues 2-pyrrolidone and pyrrolidonecarboxylate are competitive inhibitors of the enzyme. The binding of substrates and inhibitors to the active site of the enzyme is discussed.
A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme. NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.
On the basis of amino acid composition, tryptic fingerprints and the determination of amino acid sequences around the four cysteine residues, it can be concluded that chorismate mutase/prephenate dehydratase from Escherichia coli K12 consists of identical, or closely similar subunits. It follows from this that the mutase and dehydratase activities of the enzyme are probably catalysed on the one subunit.
1. 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units/mg which is about 1000 times that found in cell-free extracts of wild-tupe E. coli K12. 2. The minimum molecular weight of the enzyme was estimated by dodecylsuphate-gel electrophoresis to be 33000. Re-estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000. 3. Amino acid anaktsus abd tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical. 4. The purified enzyme does not contain Co2+.
1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.
1. Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity. Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines. D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity. 2. Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic. 3. Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM. 4. Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.
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