Evidence for an "active serine" in each fatty acid synthetase peptide.

Ultracentrifugally homogeneous fatty acid synthetase was isolated from the uropygial gland of goose by a one-step purification procedure. Formation of fatty acids from malonyl-CoA and hydrolysis of palmitoyl-CoA catalyzed by the synthetase were inhibited to an equal extent by diisopropylfluorophosphate. With labeled inhibitor, it was shown that one mole of the inhibitor was covalently attached per mole of the subunit of the enzyme. Sodium dodecyl sulphate electrophoresis showed that all of the label was contained in a 270,000 M.Wt peptide. That the active serine was not at the loading site was suggested by the observations that neither acetylation nor malonylation of the enzyme affected the reaction of the enzyme with the inhibitor and acetylation or malonylation of the enzyme was not affected by this inhibitor. Thus, each fatty acid synthetase peptide is shown to have one active serine which most probably is at the chain terminating active site of the peptide.     

Molecular basis of "suppressor" macrophages. Arginine metabolism via the nitric oxide synthetase pathway.

A molecular explanation for "suppressor" macrophage inhibition of lymphocyte proliferation is described. NG-monomethyl-L-arginine (NGMMA), a specific inhibitor of the nitric oxide synthetase pathway, markedly augments Con A-induced proliferation of rat splenic leukocytes. Macrophages are necessary and sufficient for NGMMA-releasable-suppression, as indicated by a loss of suppression after either pretreatment of isolated splenic macrophages with NGMMA or their depletion by plastic adherence or L-leucine methyl ester. L- (but not D-) arginine overrides NGMMA-releasable suppression, and suppression is blocked by RBC as would be expected if nitric oxide were the effector molecule. Unlike rats, NGMMA did not augment Con A-induced proliferation of normal mouse splenic leukocytes. However, NGMMA did augment Con A-induced proliferation of mouse splenic leukocytes induced to contain suppressor macrophages by intravenous injection of Corynebacterium parvum, which suggests a quantitative, not qualitative, difference in suppressor macrophages between rats and mice. Nitrite production, as an indicator of nitric oxide synthesis, correlated with suppressor macrophage activity in rats and mice and was inhibited by NGMMA. Finally, NGMMA also markedly enhanced proliferation with every other mitogen examined (PHA, protein A, PWM, and LPS). It is concluded that immunoregulation of lymphocyte proliferation by suppressor macrophages is mediated, in part, directly or indirectly by products of the nitric oxide synthetase pathway.     

ACV synthetase.

ACV synthetase (ACVS) is the first enzyme and plays a key role in the biosynthesis of all natural penicillins and cephalosporins. The enzyme is extremely unstable and little had been known about it until recently. This article summarizes the progress in research on this enzyme, including the establishment of a cell-free assay system, stabilization, purification, characterization, and gene cloning. A possible reaction sequence for ACVS catalysis is suggested.     

Methionyl-tRNA synthetase.

Methionyl-tRNA synthetase (MetRS) belongs to the family of 20 enzymes essential for protein biosynthesis. It links covalently methionine with its cognate tRNA. Crystal structures solved for bacterial MetRSs have given a number of interesting insights into enzyme architecture and methionylation catalysis. A comparison of sequences of MetRSs belonging to all kingdoms of life, as well as numerous biochemical and genetic studies have revealed the presence of various additional domains appended to the catalytic core of synthetase. They are responsible for interactions with tRNA and proteins. Tertiary structure of C-terminal tRNA-binding appendices can be deduced from those determined for their homologues: tRNA binding protein 111 and endothelial monocyte-activating polypeptide II. Contacts between MetRS and other proteins could be mediated not only by noncatalytic peptides but also by structural elements present in the catalytic core, e.g. Arg-Gly-Asp (RGD) motifs. Additional activities involve MetRS in the maintenance of translational fidelity and in coordination of ribosome biogenesis with protein synthesis.     

Histidyl-tRNA synthetase.

Histidyl-tRNA synthetase (HisRS) is responsible for the synthesis of histidyl-transfer RNA, which is essential for the incorporation of histidine into proteins. This amino acid has uniquely moderate basic properties and is an important group in many catalytic functions of enzymes. A compilation of currently known primary structures of HisRS shows that the subunits of these homo-dimeric enzymes consist of 420-550 amino acid residues. This represents a relatively short chain length among aminoacyl-tRNA synthetases (aaRS), whose peptide chain sizes range from about 300 to 1100 amino acid residues. The crystal structures of HisRS from two organisms and their complexes with histidine, histidyl-adenylate and histidinol with ATP have been solved. HisRS from Escherichia coli and Thermus thermophilus are very similar dimeric enzymes consisting of three domains: the N-terminal catalytic domain containing the six-stranded antiparallel beta-sheet and the three motifs characteristic of class II aaRS, a HisRS-specific helical domain inserted between motifs 2 and 3 that may contact the acceptor stem of the tRNA, and a C-terminal alpha/beta domain that may be involved in the recognition of the anticodon stem and loop of tRNA(His). The aminoacylation reaction follows the standard two-step mechanism. HisRS also belongs to the group of aaRS that can rapidly synthesize diadenosine tetraphosphate, a compound that is suspected to be involved in several regulatory mechanisms of cell metabolism. Many analogs of histidine have been tested for their properties as substrates or inhibitors of HisRS, leading to the elucidation of structure-activity relationships concerning configuration, importance of the carboxy and amino group, and the nature of the side chain. HisRS has been found to act as a particularly important antigen in autoimmune diseases such as rheumatic arthritis or myositis. Successful attempts have been made to identify epitopes responsible for the complexation with such auto-antibodies.     

Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase.

Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.     

gamma-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity.

gamma-Glutamylcysteine synthetase was purified from rat liver by an improved method involving chromatography on Sepharose-aminohexyl-ATP to a specific activity of about 1600 units/mg, or approximately twice that previously obtained; it is thus the most active preparation of this enzyme thus far isolated. The earlier preparation, which is homogeneous on polyacrylamide gel electrophoresis, exhibits "half of the sites" reactivity in that it binds a maximum of 0.5 mol of the inhibitor L-methionine-S-sulfoximine phosphate per mol of enzyme. In contrast, the present enzyme preparation binds 1 mol of methionine sulfoximine phosphate per mol of enzyme; it also differs from the enzyme obtained earlier in exhibiting much less ATPase activity and less activity in catalyzing ATP-dependent cyclization of glutamate. gamma-Glutamylcysteine synthetase dissociates in sodium dodecyl sulfate into two nonidentical subunits of apparent molecular weights 74,000 and 24,000; after cross-linking with dimethyl-suberimidate, a species having a molecular weight of about 100,000 was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. New information has been obtained about the interaction of the enzyme with glutamate analogs; thus, the enzyme is active with such glutamate analogs as beta-glutamate, N-methyl-L-glutamate, and threo-beta-hydroxy-L-glutanate, and it is effectively inhibited by cis-1-amino-1,3-dicarboxycyclonexane, 2-amino-4-phosphonobutyrate, and gamma-methylglutamate.     

The microsomal dicarboxylyl-CoA synthetase.

Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo.     

A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.     

Rabbit liver acetyl-CoA synthetase.

Acetyl-CoA synthetase (EC 6.2.1.1) was assayed in subcellular fractions of rabbit liver homogenates. The activity was located almost exclusively in the cytosol. There was no decrease in activity when butyrate or propionate (each at 5--20 mM) were added to the assay medium.     

Cysteinyl-tRNA synthetase: determination of the last E. coli aminoacyl-tRNA synthetase primary structure.

The gene coding for E. coli cysteinyl-tRNA synthetase (cysS) was isolated by complementation of a strain deficient in cysteinyl-tRNA synthetase activity at high temperature (43 degrees C). Sequencing of a 2.1 kbp DNA fragment revealed an open reading frame of 1383 bp coding for a protein of 461 amino acid residues with a Mr of 52,280, a value in close agreement with that observed for the purified protein, which behaves as a monomer. The sequence of CysRS bears the canonical His-Ile- Gly -His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS) motifs characteristic of the group of enzymes containing a Rossmann fold; furthermore, it shows striking homologies with MetRS (an homodimer of 677 residues) and to a lesser extent with Ile-, Leu-, and ValRS (monomers of 939, 860, and 951 residues respectively). With its monomeric state and smaller size, CysRS is probably more closely related to the primordial aminoacyl-tRNA synthetase from which all have diverged.     

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

The acyl-CoA synthetase "bubblegum" (lipidosin): further characterization and role in neuronal fatty acid beta-oxidation.

Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant "bubblegum," belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and beta-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells.     

Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheniformis glutamine synthetase

The relationships of five feedback inhibitors for the Bacillus licheniformis glutamine synthetase were investigated. The inhibitors were distinguishable by differences in their competitive relationship for the substrates of the enzyme. Mixtures of l-glutamine and adenosine-5'-monophosphate (AMP) or histidine and AMP caused synergistic inhibition of glutamine synthesis. Histidine, alanine, and glycine acted antagonistically toward the l-glutamine inhibition. Alanine acted antagonistically toward the glycine and histidine inhibitions. Independence of inhibitory action was observed with the other pairs of effectors. Possible mechanisms by which the inhibitors may interact to control glutamine synthesis are discussed. The low rate of catalysis of the glutamyl transfer reaction by the B. licheniformis glutamine synthetase can be attributed to the fact that l-glutamine serves both as a substrate and an inhibitor for the enzyme. Effectors which act antagonistically toward the l-glutamine inhibition stimulated glutamotransferase activity. The stimulation was not observed when d-glutamine was used as substrate for the glutamyl transfer reaction.     

Substrate selectivity of squalene synthetase.

Six 1-3H-labeled analogues of farnesyl pyrophosphate have been studied as potential substrates for yeast and rat liver squalene synthetases: 2-methylfarnesyl pyrophosphate (4), 3-demethylfarnesyl pyrophosphate (5), 7,11-dimethyl-3-ethyl-2,6,10-dodecatrienyl pyrophosphate (6), 6,7,10,11-tetrahydrofarnesyl pyrophosphate (7), 4-methylthiofarnesyl pyrophosphate (8), and 4-fluorofarnesyl pyrophosphate (9). Analogues 4 and 5 are enzymatically incorporated into 11-methylsqualene (10) and 10-demethylsqualene (11), respectively, even if no farnesyl pyrophosphate is added to the incubations. None of the other analogues gives nonpolar products with either the yeast or liver enzymes. No tritium is enzymatically released to the medium from any of the analogues, indicating that they are not accepted at the first (proton exchanging) site. The data rule out formation of dead-end presqualene pyrophosphate products with analogues as first, but not as second, substrates. Implications of these results for the enzyme active-site topology and mechanism are discussed.     

Peroxidase-dependent deactivation of prostacyclin synthetase.

A study of the enzymes of the arachidonic acid cascade revealed a high sensitivity of prostacyclin synthetase and a complete resistance of thromboxane A2 synthetase to time-dependent destruction by an oxidant [Ox] released during the peroxidase-catalyzed reduction of hydroperoxy fatty acids. The destructive action of [Ox] derived from prostaglandin G1 (PGG1), 15-hydroperoxy-PGE1, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid upon prostacyclin synthetase was prevented by 2-aminomethyl-4-t-butyl-6-iodophenol. On the other hand, deactivation resulting from PGG2 metabolism was neither time-dependent nor sensitive to 2-aminomethyl-4-t-butyl-6-iodophenol. The possibility that the action of [Ox] may alter the arachidonic acid cascade in favor of thromboxane A2 is discussed in view of its possible implications in inflammatory and other pathological processes.     

Carbohydrates in mammalian tryptophanyl-tRNA synthetase.

Homogeneous preparations of bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) contain monosaccharides (mannose, fucose, galactose, N-acetylglucosamine) as revealed by liquid chromatography. Their content comprises 2.5-3.0% (w/w) of the enzyme composed of two subunits (60 kDa x 2). The same set of sugars was detected in elastase and CNBr-generated fragments (with molecular masses of approx. 40 kDa and 30 kDa, respectively). It is concluded that bovine tryptophanyl-tRNA synthetase, in addition to being a metallo- and phosphoprotein, is also a glycoprotein.     

Characterization of three-fraction mycobacillin synthetase.

Mycobacillin synthetase lacks aspartic acid racemase, alanine racemase and glutamic acid racemase activities. The enzyme also does not respond to ATP-[32P]Pi exchange, nor does it catalyse the antibiotic synthesis in presence of amino acids of configuration opposite to that present in the molecule. Preincubation with optical isomers of opposite configuration inhibited the ATP-[32P]Pi exchange reaction to the extent of 60-90%. None of the three fractions of mycobacillin synthetase contained a pantothenic acid arm. Two molecules of ATP are required to synthesize one peptide bond of mycobacillin. Intermediate peptides of mycobacillin are not covalently linked to the three-fraction mycobacillin synthetase.