PRPP synthetase superactivity

Phosphoribosylpyrophosphate(PRPP) synthetase(PRS) catalyzes the formation of PRPP from ATP and ribose-5-phosphate. PRPP is an important substrate for the synthesis of purine, pyrimidine, and pyridine dinucleotides. Human PRS exists as complex aggregates composed of the 34 kDa catalytic subunits(PRS1 and PRS2) and other 39 kDa component designated PRPP synthetase-associated protein (PAP39). PRS superactivity is an X-chromosome linked disorder, characterized by gout and uric acid overproduction resulting from accelerated synthesis of PRPP and purine nucleotides. Among the nearly 30 affected families identified to date, there are several families in which PRS superactivity with purine nucleotide feedback resistance are associated with neurodevelopmental abnormalities in addition to hyperuricemia and gout. Different nucleotide substitutions in the PRPS1 gene encoding PRS1 have identified in six unrelated affected families with purine nucleotide feedback-resistant PRS superactivity.     

Dissociation of prelycopersene pyrophosphate synthetase from phytoene synthetase complex of tomato fruit plastids

The partially purified phytoene synthetase enzyme complex obtained from tomato fruit plastids dissociates into two or more subunit species on chromatography in low ionic strength buffer on DEAE-cellulose. One of these subunits prelycopersene pyrophosphate synthetase, has a molecular weight of approximately 40,000, whereas the phytoene synthetase complex has a molecular weight of 200,000. The prelycopersene pyrophosphate synthetase catalyzes the conversion of isopentenyl pyrophosphate to geranylgeranyl and prelycopersene pyrophosphates. The identities of these substances were established by thin layer chromatography in several solvent systems. The formation of both geranylgeranyl and prelycopersene pyrophosphates by this enzyme supports earlier results with cruder enzyme systems which suggested that these compounds are intermediates in the synthesis of phytoene.     

Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.     

Solanesyl pyrophosphate synthetase from Micrococcus lysodeikticus

Solanesyl pyrophosphate synthetase from extracts of Micrococcus lysodeikticus was purified by DEAE-Sephadex, hydroxylapatite, and Sephadex G-100 chromatography. This enzyme was found to catalyze the trans condensation of isopentenyl pyrophosphate with geranyl pyrophosphate to afford all-trans-octaprenyl (C40) and alltrans-nonaprenyl (C45) pyrophosphate without accumulation of prenyl pyrophosphate with chain length shorter than C40. all-trans-Farnesyl and all-trans-geranylgeranyl pyrophosphate also were active as cosubstrates, though they were less effective than geranyl pyrophosphate. However, neither dimethylallyl nor cis,trans,trans-geranylgeranyl pyrophosphate was active. The molecular weight of this enzyme was estimated to be 78 000 by Sephadex G-100 filtration. An enzyme preparation from young shoots of potato was found to hydrolyze the polyprenyl pyrophosphates effectively to give the corresponding prenols.     

The phenylalanyl-tRNA synthetase specifically binds DNA

BACKGROUND: Clinical trials have been performed to compare with standard heparin a once or a twice daily regimen of low-molecular-weight heparin but no direct comparison has been done between these two low-molecular-weight heparin regimens in terms of efficacy and safety with a long-term clinical evaluation. METHODS: Patients with proximal deep vein thrombosis, confirmed by venography were randomly assigned to either nadroparin (10,250 AXa IU/ml) twice daily or nadroparin (20,500 AXa IU/ml) once daily for at least 5 days. Regimens were adjusted to bodyweight. Oral anticoagulants were started on day 1 or 2 and continued for 3 months. Patients were followed up for 3 months. The composite outcome of venous thromboembolism and death possibly related to pulmonary embolism was the primary measure of efficacy. Major bleeding was the principal measure of safety. The study was designed to show equivalence between the two regimens. RESULTS: Recurrent thromboembolic events or death possibly related to pulmonary embolism were reported in 13 patients in the once daily group (4.1%) and in 24 patients of the twice daily group (7.2%): (absolute difference 3.1% in favor of the once daily regimen; 95% confidence interval -6.6%, +0.5%). Major bleeding episodes during nadroparin treatment occurred in 4 (1.3%) and 4 patients (1.2%) in the once and twice daily groups, respectively. CONCLUSIONS: A nadroparin regimen of one injection per day is at least as effective and safe as the same total daily dose divided over two injections for the treatment of acute deep vein thrombosis.     

Allosteric dominance in carbamoyl phosphate synthetase

A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed.     

L-arginine recognition by yeast arginyl-tRNA synthetase

The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 A resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of alpha-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-alpha-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNAArg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.     

Delta-Aminolevulinic acid synthetase in the heart

The regultion of cardiac delta-aminolevulinic acid synthetase activity was studied in rat heart homogenates. Optimal conditions were determined for the measurement of delta-aminolevulinic acid and an appropriate assay was established for the heart. The activity of cardiac delta-aminolevulinic acid synthetase was determined in rats either fed ad libitum or starved for 24 or 48 h. Marked decreases in delta-aminolevulinic acid synthetase activity were observed in homogenates or mitochondrial fractions prepared from hearts of fasted animals and an explanation for previous findings that the enzyme is undetectable in heart tissue is provided. Dexamethasone treatment was effective in reversing the decreases brought about by fasting but had no effect on the delta-aminolevulinic acid synthetase activity in heart homogenates from fed rats. ACTH treatment had no effect in fed or starved rats. Decreases in delta-aminolevulinic acid synthetase activity induced by fasting were not reversed in homogenates or mitochondrial preparations by succinyl-CoA-generating systems or when alpha-ketoglutarate was substituted for succinate in homogenate preparations. Cardiac delta-aminolevulinic acid dehydratase levels are not altered by fasting. Agents such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine, which produce marked increases in hepatic delta-aminolevulinic acid synthetase activity, have no effects on the activity of this enzyme in the heart.     

Human asparaginyl-tRNA synthetase: molecular cloning and the inference of the evolutionary history of Asx-tRNA synthetase family

We have cloned and sequenced a cDNA encoding human cytoplasmic asparaginyl-tRNA synthetase (AsnRS). The N-terminal appended domain of 112 amino acid represents the signature sequence for the eukaryotic AsnRS and is absent from archaebacterial or eubacterial enzymes. The canonical ortholog for AsnRS is absent from most archaebacterial and some eubacterial genomes, indicating that in those organisms, formation of asparaginyl-tRNA is independent of the enzyme. The high degree of sequence conservation among asparaginyl- and aspartyl-tRNA synthetases (AsxRS) made it possible to infer the evolutionary paths of the two enzymes. The data show the neighbor relationship between AsnRS and eubacterial aspartyl-tRNA synthetase, and support the occurrence of AsnRS early in the course of evolution, which is in contrast to the proposed late occurrence of glutaminyl-tRNA synthetase.     

Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, L-methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with Ki = 19 microM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.     

Mechanism for irreversible self-deactivation of prostaglandin synthetase

It has been shown that prostaglandin (PG) cyclooxygenase is irreversibly self-deactivated during the oxygenation of arachidonic acid (Smith, W. L., and Lands, W.E.M. (1972) Biochemistry II, 3273-3285). Using several experimental approaches and an enzyme preparation which was highly active without artificial stimulation, we have extensively investigated the mechanism of this deactivation process. During the generation of PGH2 from arachidonic acid, oxidizing equivalents were released and the reductive breakdown of PGG2 was found to deactivate the cyclooxygenase. The cyclooxygenase-catalyzed metabolism of both arachidonic acid and PGG2 generated radicals which were scavenged by phenol. Both phenol and methional (scavengers of oxygen-centered radicals) promoted the formation of PGH2 at the expense of PGG2 and increased the initial rate and the extent of reaction prior to deactivation of the cyclooxygenase. Hence, it appears that the cyclooxygenase was irreversibly self-deactivated during the formation of PGH2 from arachidonic acid, due to its oxidation by oxygen-centered radicals formed as a result of the reductive breakdown of the hydroperoxide on PGG2. Some experiments with dithiothereitol and N-ethylmaleimide suggested that the enzyme may contain a disulfide at the active site. A mechanism has been devised which accounts for the self-deactivation phenomenon, the effect of phenol and methional, the disulfide at the active site, and the pathway of substrate oxygenation.     

beta-Lactam synthetase: a new biosynthetic enzyme

The principal cause of bacterial resistance to penicillin and other beta-lactam antibiotics is the acquisition of plasmid-encoded beta-lactamases, enzymes that catalyze hydrolysis of the beta-lactam bond and render these antibiotics inactive. Clavulanic acid is a potent inhibitor of beta-lactamases and has proven clinically effective in combating resistant infections. Although clavulanic acid and penicillin share marked structural similarities, the biosyntheses of their bicyclic nuclei are wholly dissimilar. In contrast to the efficient iron-mediated oxidative cyclization of a tripeptide to isopenicillin N, the critical beta-lactam ring of clavulanic acid is demonstrated to form by intramolecular closure catalyzed by a new type of ATP/Mg2+-dependent enzyme, a beta-lactam synthetase (beta-LS). Insertional inactivation of its encoding gene in wild-type Streptomyces clavuligerus resulted in complete loss of clavulanic acid production and the accumulation of N2-(carboxyethyl)-L-arginine (CEA). Chemical complementation of this blocked mutant with authentic deoxyguanidinoproclavaminic acid (DGPC), the expected product of the beta-LS, restored clavulanic acid synthesis. Finally, overexpression of this gene gave the beta-LS, which was shown to mediate the conversion of CEA to DGPC in the presence of ATP/Mg2+. Primary amino acid sequence comparisons suggest that this mode of beta-lactam formation could be more widely spread in nature and mechanistically related to asparagine synthesis.     

Reversible denaturation of cyclosporin synthetase by urea

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.     

Argininosuccinate synthetase activity in cultured human lymphocytes

The activity of argininosuccinate synthetase (E.C. 6.3.4.5), a urea cycle enzyme, was measured in cultured human lymphocytes using a new radioactive assay. Control cells had a maximum specific activity of 15.7 +/- 8.7 nmoles per hour per milligram of protein and an apparent Km for citrulline of 2 X 10(-4) M, whereas cells derived from a patient with citrullinemia had no detectable activity. A nutritional variant, selected out of the citrullinemic lymphocyte population by ability to grow in citrulline, had a maximum specific activity of 10.7 +/- 3.8 nmoles/hr/mg and an apparent Km for citrulline of 2 X 10(-2) M. These measurements confirm the observation that citrullinemia is associated with a defect in argininosuccinate synthetase activity and provide further evidence that citrullinemia is expressed in cultured lymphocytes. The emergence of a nutritional variant with a partial defect in argininosuccinate synthetase enzyme suggests that this citrullinemic patient has a heterogeneous population of cells, some totally defective and others only partially defective in argininosuccinate synthetase. The new activity assay is described in detail.     

Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis

In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis. Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector. A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation. We describe the key experiments leading to these conclusions.     

Prediction of substrate-specific pockets in cyclosporin synthetase

Amino acid sequence comparisons between domains of cyclosporin synthetase have been used to identify regions of the sequence which are responsible for the recognition and binding of the individual amino acids. Using a limited set of selection rules it was possible to identify three amino acid positions in the subdomain sequences which are responsible for amino acid specificity. Homology with the firefly luciferase protein shows that these three key residues are close to each other and line the surface of a putative specific substrate binding pocket located on the amino acyl-adenylation subdomain. These results allow us to predict a large number of cyclosporin synthetase mutants which could be used to synthesise alternative cyclosporin-like peptides.     

Electrostatic potential in aminoacylation by aspartyl-tRNAs synthetase

Based upon the X-ray structures of complexes between tRNAAsp and aspRS including ATP or Asp-AMP, several electrostatic potentials were calculated by solving the Poisson-Boltzmann equation. The potentials indicate clearly that a Mg2+ ion is essential for binding of ATP and that aspartate is identified electrostatically. The alpha-carboxyl group is forced to contact with the alpha-phosphorus atom of ATP, suggesting its inversion to form an Asp-AMP. When the cognate tRNA is bound to the aspRS:Asp-AMP complex, the 3'-hydroxyl group is located in an electrostatically favorable position to transfer the amino acid as a class II aminoacylation.