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Biomedical subjects

E E Snell

Publications and source records attributed to E E Snell.

At least 55 records · Page 3Linked to original sources
PubMed1983

The bacterial oxidation of vitamin B6. 4-Pyridoxic acid dehydrogenase: a membrane-bound enzyme from Pseudomonas MA-1.

A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.

Alcohol Oxidoreductases↗
PubMed1983

Phosphorus 31 nuclear magnetic resonance study of tryptophanase. Pyridoxal phosphate-binding site.

The pyridoxal phosphate-dependent enzyme tryptophanase has been investigated using 31P nuclear magnetic resonance at 72.86 MHz. In the native enzyme, the pyridoxal-P 31P chemical shift was found to be 3.55 ppm and independent of pH, indicating that the dianionic phosphate group of the cofactor is not accessible to solvent. Binding of the competitive inhibitor, beta-phenyl-DL-serine, results in the formation of the transaldimination complex. This complex is fixed to the enzyme via the dianionic phosphate group of the cofactor: again the observed shift is independent of pH. In both cases, restricted rotational freedom of the phosphate group around the C-O bond linking the phosphate ester to the pyridine moiety of the cofactor could be asserted from line width data. Addition of the competitive inhibitor, L-alanine, to tryptophanase produces the quinonoid intermediate. The phosphate group of this complex has lost its specific interaction (probably a salt bridge) with the protein, as indicated by the pH dependence of the chemical shift.

Binding Sites↗
PubMed1983

Pyruvoyl-dependent histidine decarboxylases from Clostridium perfringens and Lactobacillus buchneri. Comparative structures and properties.

Histidine decarboxylase (EC 4.1.1.22) was purified to homogeneity from Clostridium perfringens and also from Lactobacillus buchneri. Both enzymes are composed of alpha and beta subunits, with an essential pyruvoyl group bound to the alpha subunit. In this respect and also in molecular weight of both the alpha and beta subunits and the native enzyme, they closely resemble the previously described (Riley, W.D., and Snell, E. E. (1970) Biochemistry 9, 1485-1491) histidine decarboxylase from Lactobacillus 30a. Rabbit antibodies to the latter enzyme cross-react incompletely with the decarboxylase from L. buchneri but not with that from C. perfringens in double diffusion tests. The clostridial decarboxylase differs substantially from the Lactobacillus 30a enzyme in amino acid composition and, unlike the latter enzyme, requires high ionic strength (I approximately 1.4 M) for maximum activity. The enzymes also differ in rates of electrophoretic migration. A proenzyme for the decarboxylase similar to that previously found in Lactobacillus 30a was detected in immunoprecipitates of extracts of L. buchneri. We conclude that these proteins arise from pyruvate-free precursor proteins by similar mechanisms and probably have diverged from a common ancestral protein.

Amino Acids↗
PubMed1983

Conversion of prohistidine decarboxylase to histidine decarboxylase: peptide chain cleavage by nonhydrolytic serinolysis.

Unlabeled prohistidine decarboxylase and prohistidine decarboxylase containing L-[carboxyl-(18)O]serine or L-[hydroxyl-(18)O]serine were isolated in homogeneous form from mutant 3 of Lactobacillus 30a grown with the appropriately labeled serine. There was no randomization or redistribution of label during growth, isolation of the protein, or enzymatic hydrolysis and reisolation of the labeled amino acids. These proteins were used to show that during proenzyme activation, in which individual pi subunits of the proenzyme are converted to alpha and beta subunits of the active enzyme [Formula: see text] (in which pi, alpha, and beta subunits have the partial structures shown and Prv designates a pyruvoyl group), no (18)O from H(2) (18)O is incorporated into the newly formed carboxyl terminus (Ser-81) of the beta chain, although no labilization of (18)O from proenzyme labeled with L-[carboxyl-(18)O]serine occurred when the proenzyme was activated in H(2) (16)O by the same procedures. The additional oxygen atom present in the carboxyl group of Ser-81 of the beta subunit is transferred from the hydroxyl group of Ser-82 of the proenzyme during the activation reaction. The same result was obtained with wild-type enzyme formed intracellularly. Peptide bond cleavage during activation of the proenzyme thus proceeds by a hitherto unobserved direct or indirect "serinolysis" coupled to alpha,beta-elimination at Ser-82 to yield the pyruvoyl group of the alpha subunit, rather than by hydrolysis. Possible mechanisms for the reaction are discussed briefly.

Amino Acid Sequence↗
PubMed1982

Histidine decarboxylase of Lactobacillus 30a. Comparative sequences of the beta chain from wild type and mutant enzymes.

Manual and automated sequencing of peptides isolated from tryptic, chymotryptic, and Staphylococcus aureus V8 protease digests of the beta chain of histidine decarboxylase from Lactobacillus 30a have established the following sequence for the wild type protein: NH2-Ser-Gly-Leu-Asp-Ala-Lys-Leu-Asn-Lys-Leu-Gly-Val-Asp-Arg-Ile-Ala-Ile-Ser-Pro -Tyr-Lys-Gln-Trp-Thr-Arg-Gly-Tyr-Met-Glu-Pro-Gly-Asn-Ile-Gly-Asn-Gly-Tyr-Val-Thr-Gly-Leu-Lys-Val-Asp-Ala-Gly-Val-Arg-Asp-Lys-Ser-Asp-Asp-Asp-Val-Leu-Asp-Gly-I le-Val-Ser-Tyr-Asp-Arg-Ala-Glu-Thr-Lys-Asn-Ala-Tyr-Ile-Gly-Gln-Ile-Asn-Met-Thr- Thr-Ala-Ser-COOH The beta chain from mutant 3 of this organism shows two amino acid replacements: Ser-51 is replaced by Ala and Gly-58 by Asp. These amino acid replacements result in a significant increase in the predicted alpha-helical content and a significant decrease in the isoelectric point of the mutant beta chain and are consistent with changes in physical and catalytic properties of the mutant histidine decarboxylase. In addition, about 15% of the mutant chains contain Met-Ser at the NH2 terminus rather than Ser. Asn-35 is partially deamidated in both proteins. Structural comparisons show that the histidine decarboxylase from Lactobacillus 30a and a similar pyruvoyl enzyme from Micrococcus sp. n. have evolved from a common ancestral protein.

Amino Acid Sequence↗
PubMed1981

Kinetic investigations on a flavoprotein oxygenase, 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

In the presence of NADH and O2, 2-methyl-3-hydroxypyridine-5-carboxylate oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 catalyzes reductive oxygenation of 2-methyl-3-hydroxypyridine-5-carboxylate (Cpd I) to yield alpha-N-(acetylaminomethylene)succinic acid (Cpd A). Steady state kinetic data and studies with alternate substrates are consistent only with an ordered mechanism in which Cpd I binds first, followed by NADH; the first product, NAD+, is then released. This event is followed by oxygen binding, and finally release of the oxygenated and reduced cleavage product, Cpd A. This kinetic mechanism was confirmed by studying inhibition by NAD+, which binds competitively with oxygen, but not with NADH. The kinetic mechanism of this reaction resembles that proposed for bacterial flavin monooxygenases that catalyze hydroxylation of aromatic homocyclic compounds.

Flavoproteins↗
PubMed1981

Crystallization and subunit structure of histidine decarboxylase from Lactobacillus 30a.

Histidine decarboxylase from Lactobacillus 30a has been crystallized in a variety of forms which together indicate a revised subunit structure for the native particle. Octahedral crystals of the wild type enzyme obtained at room temperature from ammonium sulfate solutions in microdiffusion cells belong to tetragonal space group I4122 with a = b = 222 A and c = 107.5 A. Trigonal and hexagonal plates of prohistidine decarboxylase and activated proenzyme obtained at 4 degrees C from polyethyleneglycol solutions by vapor equilibration using the hanging drop technique belong to the trigonal space group P321 with a = b = 100 A and c = 164 A. The space group symmetries and unit cell contents of these crystals indicate 32 point group symmetry for the subunit structure of these enzymes. Sedimentation coefficients of wild type enzyme measured as a function of ionic strength at pH 7.0 indicate a rapid equilibrium between species varying from 6.9 S to 9.4 S. Sedimentation equilibrium analysis demonstrated the existence of a nearly homogeneous particle with Mr congruent to 208,000 at ionic strengths above I = 0.20, while an additional species of approximately one-half that molecular weight is observed at very ionic strengths (I = 0.2). At the pH optimum of the enzyme (pH 4.8), te larger species is dominant at all ionic strengths tested. Electron micrographs of native wild type enzyme show a dominant tetrahedral particle approximately 60 A on an edge while similar micrographs of enzyme cross-linked with glutaraldehyde show a dumbbell-shaped particle approximately 60 A in width and 120 A in length. These results establish that: (a) the native enzyme has a Mr congruent to 208,000 and a subunit composition (alpha beta)6; (b) the proenzyme has a subunit composition (pi)6; and (c) stable (alpha beta)3 and (pi) 3 particles exist under certain conditions.

Carboxy-Lyases↗
PubMed1980

Some effects of indole on the interaction of amino acids with tryptophanase.

Although indole is a potent inhibitor (KI = 0.01 mM) of pyruvate formation from substrates of tryptophanase (EC 4.1.99.1, from Escherichia coli), we could not detect binding of indole to free tryptophanase (KD greater than 1.0 mM). However, indole, skatole, and toluene increased the affinity of tryptophanase for certain inhibitory amino acids. Binding of amino acids with small side chains (e.g. Ala, Gly) was increased, but there was little or no effect on the binding of amino acids with bulky side chains (e.g. norvaline, ethionine). These effects were quantitated by using changes in the absorption spectra of the enzyme . amino acid complexes. Indole decreases the absorbance obtainable at 500 nm for amino acids with small hydrophobic side chains (L-Ala, Gly), increases this absorbance for amino acids with small polar side chains (beta-cyano-L-alanine), and does not change the spectra of tryptophanase complexes with amino acids with bulky side chains, i.e. amino acids whose binding affinities are unaffected by indole. These spectral differences are interpreted in terms of an effect of bound indole (or side chain binding) on the partitioning of the bound amino acid between catalytic forms of the enzyme. The data indicate that substrate-induced conformational changes occur at the enzyme active site that generate a high affinity indole-binding site during catalytic turnover of tryptophanase and are important in the catalytic functioning of the enzyme. These changes also explain reproducible differences in KI values observed previously for amino acids in different assay systems used for steady state kinetic inhibition studies. The optimal conditions for the growth of E. coli for tryptophanase production are outlined, together with a procedure for purification of holotryptophanase.

Alanine↗
PubMed1980

Purification and properties of ornithine decarboxylase from Lactobacillus sp. 30a.

Inducible ornithine decarboxylase from Lactobacillus sp. 30a has been purified to homogeneity as judged by ultracentrifugation and gel electrophoresis. Unlike histidine decarboxylase from the same species (a pyruvoyl enzyme), ornithine decarboxylase is a pyridoxal phosphate enzyme. The purified enzyme is specific for L-pornithine (Km 1.7 mM; specific activity, 150 to 200 mumol min-1 mg-1 at 37 degrees C) and is inhibited by various homologous omega-amino acids, amines, and polyamines. The native enzyme has an isoelectric point of 4.55 and a molecular weight of 1.04 X 10(6). At pH 7.3 and above, it dissociates reversibly to a species of Mr = 184,000, and on gel electrophoresis in the presence of sodium dodecyl sulfate shows a single band of Mr = 85,000. We ascribe these species to the dodecamer, dimer, and monomer, respectively, of a single peptide subunit; electron micrographs show a hexagonal array of apparently dimeric subunits in the native enzyme. Highest enzymatic activity is present in the dodecamer. The holoenzyme is resolved by dialysis against cysteine; spectrophotoemetric titration of the apoenzyme with pyridoxal 5'-phosphate indicates the presence of 1 coenzyme-binding site/monomeric subunit.

Carboxy-Lyases↗
PubMed1979

Isolation of temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium and mapping of the coaA gene.

Temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium LT2 were selected by using the excretion of pantothenate at the nonpermissive temperature as a screening method. Thermolability of the pathothenate kinase activity in extracts of the mutants was demonstrated. The mutations were mapped at min 89 of the Salmonella chromosome, near rpoB, by transduction. As pantothenate kinase catalyzes the first step in the biosynthesis of coenzyme A from pantothenate, the new genetic locus has been designated coaA.

Cell-Free System↗