Subunit interactions in aspartate transcarbamylase. The interaction between catalytic and regulatory subunits and the effect of ligands.
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The interaction between the catalytic subunit (c3) and the regulatory subunit (r2) of aspartate transcarbamylase from Escherichia coli was studied by measuring the reversible formation of the c3r6 complex as a function of r2 concentration. Conversion to the native enzyme was prevented by using a very low concentration of c2 (40 ng per ml) in the presence of bovine serum albumin. A simple hyperbolic r2 saturation curve was obtained suggesting the presence of only one kind of c:r domain. From the association constant for the formation of c3r6, the free energy of c:r interaction can be estimated to be about -10 Cal per mole. Neither CTP nor ATP appears to affect the strength of c:r interaction in this complex. Succinate in the presence of carbamyl phosphate promotes tighter binding. At higher concentration of c3 and nonsaturating levels of r2, conversion to the native enzyme (c3r6) takes place. This renaturation process is second order with respect to the concentration of c3 and is virtually irreversible. Renaturation is inhibited by saturating levels of r2 and to some extent by both CTP and ATP. The effect of ligands on c:r interactions reported here may have significance in the allosteric mechanism of the native enzyme. (+info)
Subunit interactions in aspartate transcarbamylase. A model for the allosteric mechanism.
(34/1470)
The conformational changes in aspartate transcarbamylase upon binding of substrates or regulatory ligands and the effects of alterations in the subunit structure on the allosteric interactions are reviewed. The available information including recent results from studies of the c3r6 complex (c denotes the catalytic polypeptide and r, the regulatory polypeptide) is considered in terms of the existing models for the discrepancies between experimental observations and the present models could be resolved by postulating an important role for r:r interactions in the allosteric mechanism. A new model is presented in which an obligatory conformational change upon binding of substrates results in an alteration in the relative orientation of c versus r. As a consequence of symmetry conservation, the r:r domain is shifted to a position of higher potential energy. By favoring one or the other alternative r:r domains, CTP and ATP can respectively enhance and reduce the sigmoidal character of substrate saturation. The model is shown to be consistent with all of the important known properties of the enzyme. Because the heterotropic effects of CTP or ATP are postulated to operate via a mechanism separate from that for the homotropic effects of the substrates, this model accounts satisfactorily for the observation by Kerbiriou and Herve (Kerbiriou, D., and Herve, G. (1973) J. Mol. Biol. 78, 687-702) that homotropic effects can be abolished whereas heterotropic effects are retained in the altered enzyme from Escherichia coli grown in the presence of 2-thiouracil. (+info)
Characterization of a sulfur-regulated oxygenative alkylsulfatase from Pseudomonas putida S-313.
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The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the alpha-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C(4) to C(12), required oxygen and Fe(2+) for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized alpha-ketoglutarate-dependent dioxygenases, AtsK accepted a range of alpha-keto acids as co-substrates, including alpha-ketoglutarate (K(m) 140 microm), alpha-ketoadipate, alpha-ketovalerate, and alpha-ketooctanoate. The measured K(m) values for hexyl sulfate and SDS were 40 and 34 microm, respectively. The apparent M(r) of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the alpha-ketoglutarate-dependent dioxygenase family. (+info)
Analysis of glutamate exit in Escherichia coli.
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Escherichia coli B exhibits carrier-mediated first-order exit of glutamate with a half-time of less than 4 min, similar to that observed in K-12 strains. Glutamate exit in both B and K-12 strains is inhibited by arsenite. Practically all of the radioactivity lost during exit by K-12 cells has been accounted for as glutamate in the cell filtrate. (+info)
Variation of ribosomal proteins with bacterial growth rate.
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The composition of ribosomal proteins has been examined as a function of the growth rate of Escherichia coli cells. Seven sets of cultural conditions, utilizing different combinations of carbon and nitrogen sources, were employed to provide a 36-fold spread in growth rate. The cellular content of most of the ribosomal proteins in ribosomes decreased to a similar extent in the very slow-growing cultures. Major exceptions were proteins S6 and L12, which exhibited a much more pronounced decrease , and S21, which exhibited an increase. None of the proteins remained invariant with growth rate. (+info)
Effects of colicin Ia on transport and respiration in Escherichia coli.
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Treatment of Escherichia coli with colicin Ia leads to an inhibition in the active transport of exogenously supplied proline, thiomethyl-beta-D-galactoside and potassium ion. Furthermore, the addition of colicin Ia to cells preloaded with these substances leads to their almost immediate efflux. In contrast, colicin tia treatment enhances by as much as 10-fold the level of accumulation of alpha-methyl-D-glucoside. The colicin Ia-induced stimulation of glucoside accumulation is mediated by the phosphotransferase system. Cells treated with colicin Ia exhibit an increased rate of respiration when glucose is the substrate and a decreased rate when glycerol or succinate is the substrate; The decreased rate of succinate-dependent respiration is probably due to the failure of Ia-treated cells to accumulate succinate. (+info)
Purification and properties of a periplasmic glutamate-aspartate binding protein from Escherichia coli K12 strain W3092.
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A protein which binds both glutamate (K-D = 0.8 muM) and aspartate (K-D = 1.2 muM) has been purified to homogeneity (290-fold) from the periplasmic fraction released from Escherichia coli W3092 by the cold osmotic shock procedure. The apparent molecular weight of the glutamate-aspartate binding protein is approximately 31,000 as judged by gel electrophoresis, gel filtration, and sedimentation equilibrium centrifugation; and the protein has a pI of 9.69. This protein contains 2 half-cystine residues and is dependent on a dithiothreitol-sensitive component for renaturation to an active conformation following urea or guanidine treatment. Of the natural amino acids only the L isomers of glutamate, aspartate, glutamine, asparagine, and alanine were inhibitors of either [C]glutamate or [14C]aspartate binding and the inhibitions were competitive. Only one binding site is indicated per molecule of protein. Antibody prepared against the glutamate-asparate binding protein does not cross-react with purified glutamine binding protein or any other component of osmotic shock fluid. The antibody does cross-react with osmotic shock fluids obtained from E. coli strains B and W and Salmonella typhimurium OT2. The glutamate-aspartate binding protein-antibody complex does not bind either glutamate or aspartate. The protein may be similar to the glutamate binding activity detected in the periplasmic fraction released from E. coli strain B (Miner, K.M., and Frank, L. (1974) J. Bacteriol. 117, 1093-1098) and strain K12 CS (Barash, H., and Halpern, Y.S. (1971) Biochem. Biophys. Res. Commun. 45, 681-688). This protein appears to function in the transport of glutamate by E. coli strain W cultured in minimal medium with succinate as the carbon source (Willis, R.C., and Furlong, C.E. (1975) J. Biol. Chem. 250, 2581-2586. (+info)
Cross-linking of Escherichia coli succinic thiokinase. I. Reaction with diiminoesters and dimaleimides.
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Treatment of the tetrameric alpha2 beta2 protein succinic thiokinase of Escherichia coli with dimethylsuberimidate (DMS) yielded five protein species detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These five protein species had estimated molecular weight values of 29,500, 41,000, 73,000, 117,000, and 132,000, and corresponded to alpha monomer, beta monomer, alpha beta dimer, alpha2 beta trimer and alpha2 beta2 tetramer, respectively. In all cases, the cross-linking produced predominantly the 73,000 molecular weight dimer with respectively lesser amounts of the tetramer and trimer. Succinic thiokinase was also cross-linked by reaction with N, N'-o-phynylenedimaleimide or with N, N'-P-phenylenedimaleimide. In these instances, treatment produced the alpha beta dimer as the only oligomeric species. Ammonolysis of isolated tetramer, trimer, and dimer produced by DMS treatment gave the 29,500 molecular weight monomer (alpha monomer) and the 38,500 molecular weight monomer (beta monomer). The absence of dimers of like subunits and the predominance of the dimer of unlike subunits are consistent with a quaternary structure of the native enzyme in which unlike subunits are closely associated but like subunits are not. Under certain conditions, an additional dimer of approximately 60,000 molecular weight was produced. This appeared to result from cleavage of the beta chain of an alpha beta dimer. Phosphorylation of native succinic thiokinase with [gamma-32P]ATP and [gamma-32P]GTP showed radioactivity only in the alpha monomer. Phosphorylation of enzyme before or after cross-linking showed radioactivity in all cross-linked bands except the beta monomer. Experiments in which the enzyme was titrated with [14C]DMS and trinitrobenzenesulfonate revealed that approximately half of the available amino groups reacted with the diiminoester, but that a small fraction of these (smaller than 20%) had reacted bifunctionally (+info)