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H R Levy

Publications and source records attributed to H R Levy.

At least 55 records · Page 3Linked to original sources
PubMed1976

Anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferases from Salmonella typhimurium. Purification of the enzyme complex and analysis of multiple forms.

1. The anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme complex (chorismate pyruvatelyase (amino-accepting), EC 4.1.3.27) - (N-(5'-phosphoribosyl)-anthranilate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.18), from Salmonella typhimurium has been purified with high yields to homogeneity. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme complex revealed one major band containing 96% of the protein. The final yield of enzyme complex activity ranged from 30 to 60%. The absorbance spectrum of enzyme complex showed a peak at 280 nm and fine structure with peaks at 253, 259, 266 and 269 nm. These latter wavelengths correspond closely with the known absorbance maxima of phenylalanine. 2. When purified enzyme complex was subjected to standard gel electrophoresis, a four band pattern of protein peaks was consistently observed. The major enzyme complex band was apparently the native tetramer, having a molecular weight 280 000 and containing ammonia- and glutamine-dependent anthranilate synthase activity. The other three bands were molecular weight isomers of the major enzyme complex band. Two forms of molecular weight isomers were present: dimers and an aggregate of the native enzyme complex. The molecular weight isomers of the enzyme complex may represent forms generated by aggregation and denaturation of the native enzyme complex. 3. A new and highly sensitive spectrophotometric assay for phosphoribosyl-transferase is described. The method is based upon the difference in extinction coefficients between anthranilate and N-(5'-phosphoribosyl)anthranilate.

Anthranilate Phosphoribosyltransferase↗
PubMed1976

Metal ion requirement and tryptophan inhibition of normal and variant anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase complexes from Salmonella tyrhimrium.

1. Both Mn2+ and Co2+ can replace Mg2+ as the required divalent cation for all activities of the enzyme complex between anthranilate synthase (chorismate pyruvate-lyase (amino-accepting), EC 4.1.3.27) and anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (N(5'-phosphoribosyl)-anthranilate:pyrophosphate phosphoribosytransferase, EC 2.4.2.18) from Salmonella typhimurium. They have much lower apparent Km values than Mg2+, both for glutamine-dependent anthranilate synthase (Mn2+ = 1.1 muM, Co2+ - 2.6 muM, Mg2+ = 83 muM) and for phosphoribosyltransferase (Mn2+ = 16 muM, Co2+ = 14.6 muM, Mg2+ = 133 muM). The ratio of total Mg2+ to total Mn2+ found in a cell extract of S. typhimurium trpE2 , the source of normal enzyme complex, was found to be 350, suggesting that Mg2+ is probably utilized by the enzyme complex in vivo under our growth conditions. 2. An enzyme complex has been isolated from a mutant strain of S. typhimurium (SO-515) that has a variation in the anthranilate synthase subunit which is thought to be a single amino acid substitution. This variation causes glutamine-dependent anthranilate synthase to be hypersensitive to feedback inhibition by tryptophan (Ki = 0.4 muM compared to Ki = 20 muM for normal enzyme complex). The phosphoribosyltransferase in the variant enzyme complex is also hypersensitive to tryptophan but the kinetics are complex and involve activation by tryptophan in the presence of low amounts of 5-phosphoribosyl 1-pyrophosphate. 3. In the variant enzyme complex the apparent Km for Mg2+ is elevated to 360 muM for glutamine-linked anthranilate synthase but reduced to 75 muM for phosphoribosyltransferase. 4. These results suggest that the variant enzyme complex has altered tertiary and quaternary structures and that regulation of both activities is effected by tryptophan binding to only anthranilate synthase.

Anthranilate Phosphoribosyltransferase↗
PubMed1975

Anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase from Salmonella typhimurium. Inactivation of glutamine-dependent anthranilate synthetase by agarose-bound anthranilate.

Exposure of the anthranilate synthetase-anthranilate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (amino-accepting) EC 4.1.3.27 and N-(5-phosphoribosyl)-anthranilate pyrophosphate phosphoribosyl-transferase, EC 2.4.2.18) from Salmonella typhimurium to agarose-bound anthranilate led to the slow inactivation of glutamine-dependent anthranilate synthetase activity, an activity dependent on protein-protein interaction in the enzyme complex. Region I of phosphoribosyltransferase, the location of the enzyme complex glutaminase activity, is the site of alteration. Phosphoribosyltransferase and NH3-dependent anthranilate synthetase activities and trypto phan regulation of phosphoribosyltransferase were unaffected by the anthranilate matrix. The molecular weight (280 000) and subunit molecular weight (62 000) of the enzyme complex eluted from an anthranilate matrix were identical to those of enzyme complex purified by classical methodology. The enzyme complex could be partially protected against inactivation by storiing in 0.1 M L-glutamine or 30% glycerol and completely protected by storing in 50% glycerol at -18 degrees C. Evidence is presented that the anthranilate matrix acts as a hydrophobic matrix and may be binding to and altering a hydrophobic region in the enzyme complex. The anthranilate matrix provides a convenient tool for altering a specific region of an enzyme complex without covalent modification. At the same time, the results demonstrate that affinity matrices are not necessarily innocuous but may subject macromolecules to an adverse environment not previously recognized.

Allosteric Site↗
PubMed1975

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.

Animals↗
PubMed1975

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.

Binding Sites↗
PubMed1970

Rat mammary-gland acetyl-coenzyme A carboxylase interaction with milk fatty acids.

1. Highly purified rat mammary-gland acetyl-CoA carboxylase was inhibited by milk obtained from rats 12h after their young were weaned. 2. All the inhibitory activity was found in the particulate fraction (R(105)) obtained on centrifuging the milk. It could be extracted from milk fraction R(105) with acetone and identified as a complex mixture of non-esterified fatty acids, present in high concentration (nearly 10mm) in the milk. 3. Inhibition of acetyl-CoA carboxylase was observed at low concentrations (0.2-20mum) of several of these fatty acids when fresh fully active enzyme was used. Enzyme that had been partly inactivated by aging, or by storing in the absence of citrate, was stimulated by low concentrations but inhibited by high concentrations of fatty acids. 4. Various experiments suggested that fatty acids produce irreversible inactivation of acetyl-CoA carboxylase. 5. The effects of palmitoyl-CoA on mammary-gland acetyl-CoA carboxylase were found to resemble those of fatty acids, except that palmitoyl-CoA was effective at lower concentration. 6. The effect of milk fraction R(105) was tested on six other enzymes previously shown to decline to various extents after weaning. Although several of these enzymes were affected by unfractionated milk fraction R(105), none was significantly inhibited by the acetone extract or by low concentrations of lauric acid. 7. The findings are consistent, both qualitatively and quantitatively, with a regulatory mechanism whereby milk fatty acids shut off fatty acid synthesis in the mammary gland after weaning by inhibiting acetyl-CoA carboxylase.

Acetone↗