Kinetic mechanism of Escherichia coli asparagine synthetase B

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.     

A detailed structural description of Escherichia coli succinyl-CoA synthetase

Succinyl-CoA synthetase (SCS) carries out the substrate-level phosphorylation of GDP or ADP in the citric acid cycle. A molecular model of the enzyme from Escherichia coli, crystallized in the presence of CoA, has been refined against data collected to 2.3 A resolution. The crystals are of space group P4322, having unit cell dimensions a=b=98.68 A, c=403.76 A and the data set includes the data measured from 23 crystals. E. coli SCS is an (alphabeta)2-tetramer; there are two copies of each subunit in the asymmetric unit of the crystals. The crystal packing leaves two choices for which pair of alphabeta-dimers form the physiologically relevant tetramer. The copies of the alphabeta-dimer are similar, each having one active site where the phosphorylated histidine residue and the thiol group of CoA are found. CoA is bound in an extended conformation to the nucleotide-binding motif in the N-terminal domain of the alpha-subunit. The phosphoryl group of the phosphorylated histidine residue is positioned at the amino termini of two alpha-helices, one from the C-terminal domain of the alpha-subunit and the other from the C-terminal domain of the beta-subunit. These two domains have similar topologies, despite only 14 % sequence identity. By analogy to other nucleotide-binding proteins, the binding site for the nucleotide may reside in the N-terminal domain of the beta-subunit. If this is so, the catalytic histidine residue would have to move about 35 A to react with the nucleotide.     

Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and Pi are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.     

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.     

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.     

X-ray crystal structure of glycinamide ribonucleotide synthetase from Escherichia coli

Glycinamide ribonucleotide synthetase (GAR-syn) catalyzes the second step of the de novo purine biosynthetic pathway; the conversion of phosphoribosylamine, glycine, and ATP to glycinamide ribonucleotide (GAR), ADP, and Pi. GAR-syn containing an N-terminal polyhistidine tag was expressed as the SeMet incorporated protein for crystallographic studies. In addition, the protein as isolated contains a Pro294Leu mutation. This protein was crystallized, and the structure solved using multiple-wavelength anomalous diffraction (MAD) phase determination and refined to 1.6 A resolution. GAR-syn adopts an alpha/beta structure that consists of four domains labeled N, A, B, and C. The N, A, and C domains are clustered to form a large central core structure whereas the smaller B domain is extended outward. Two hinge regions, which might readily facilitate interdomain movement, connect the B domain and the main core. A search of structural databases showed that the structure of GAR-syn is similar to D-alanine:D-alanine ligase, biotin carboxylase, and glutathione synthetase, despite low sequence similarity. These four enzymes all utilize similar ATP-dependent catalytic mechanisms even though they catalyze different chemical reactions. Another ATP-binding enzyme with low sequence similarity but unknown function, synapsin Ia, was also found to share high structural similarity with GAR-syn. Interestingly, the GAR-syn N domain shows similarity to the N-terminal region of glycinamide ribonucleotide transformylase and several dinucleotide-dependent dehydrogenases. Models of ADP and GAR binding were generated based on structure and sequence homology. On the basis of these models, the active site lies in a cleft between the large domain and the extended B domain. Most of the residues that facilitate ATP binding belong to the A or B domains. The N and C domains appear to be largely responsible for substrate specificity. The structure of GAR-syn allows modeling studies of possible channeling complexes with PPRP amidotransferase.     

Isotope exchange studies on the Escherichia coli selenophosphate synthetase mechanism

Selenophosphate synthetase, the Escherichia coli selD gene product, is a 37-kDa protein that catalyzes the synthesis of selenophosphate from ATP and selenide. In the absence of selenide, ATP is converted quantitatively to AMP and two orthophosphates in a very slow partial reaction. A monophosphorylated enzyme derivative containing the gamma-phosphoryl group of ATP has been implicated as an intermediate from the results of positional isotope exchange studies. Conservation of the phosphate bond energy in the final selenophosphate product is indicated by its ability to phosphorylate alcohols and amines to form O-phosphoryl- and N-phosphoryl-derivatives. To further probe the mechanism of action of selenophosphate synthetase, isotope exchange studies with [8-14C]ADP or [8-14C]AMP and unlabeled ATP were carried out, and 31P NMR analysis of reaction mixtures enriched in H218O was performed. A slow enzyme-catalyzed exchange of ADP with ATP observed in the absence of selenide implies the existence of a phosphorylated enzyme and further supports an intermediary role of ADP in the reaction. Under these conditions ADP is slowly converted to AMP. Incorporation of 18O from H218O exclusively into orthophosphate in the overall selenide-dependent reaction indicates that the beta-phosphoryl group of the enzyme-bound ADP is attacked by water with liberation of orthophosphate and formation of AMP. Based on these results and the failure of the enzyme to catalyze an exchange of labeled AMP with ATP, the existence of a pyrophosphorylated enzyme intermediate that was postulated earlier can be excluded.     

ATP-dependent inactivation of Escherichia coli gamma-glutamylcysteine synthetase by L-glutamic acid gamma-monohydroxamate

Incubation of Escherichia coli gamma-glutamylcysteine synthetase with L-glutamic acid gamma-monohydroxamate and ATP caused slow but irreversible inhibition of the enzyme, and more than 90% activity was lost in three days. The enzyme was not inactivated when ATP was absent or L-aspartic acid beta-monohydroxamate was substituted for L-glutamic acid gamma-monohydroxamate, suggesting that the inactivation process reflected a mechanism-based reaction of L-glutamic acid gamma-monohydroxamate and ATP.     

Pantothenate synthetase of Escherichia coli B. I. Physicochemical properties

Using a new apparatus for preparative polyacrylamide gel electrophoresis, pantothenate synthetase (D-pantoate: beta-alanine ligase (AMP-forming), [EC 6.3.2.1] was purified about 500-fold from Escherichia coli B. It was found to be homogeneous in analytical disc gel electrophoresis and sedimentation ultracentrifugation (so20, w=4.9). From sedimentation equilibrium ultracentrifugation, a molecular weight of 70,100 was obtained, which is in good agreement with the value obtained by the Sephadex G-150 gel filtration method (69,000); the diffusion constant was calculated to be 5.88X10(-7) cm2/sec. The minimum molecular weight calculated from the amino acid composition of this enzyme protein was 19,700, a value in reasonable accord with molecular weight of the enzyme subunit, 18,000, obtained by gel electrophoresis in the presence of sodium dodecylsulfate. The partial specific volume, v, was calculated to be 0.71 cm3/g. The enzyme had an amino-terminal glycyl residue and a Leu-Ala-Ser-OH sequence at the carboxyl end. Electrophoresis of the enzyme with carrier ampholine gave an isoelectric point of pH 4.6.     

Refined crystal structures of guanine nucleotide complexes of adenylosuccinate synthetase from Escherichia coli

Structures of adenylosuccinate synthetase from Escherichia coli complexed with guanosine-5'-(beta,gamma-imido) triphosphate and guanosine-5'-(beta,gamma-methylene)triphosphate in the presence and the absence of Mg2+ have been refined to R-factors below 0.2 against data to a nominal resolution of 2.7 A. Asp333 of the synthetase hydrogen bonds to the exocyclic 2-amino and endocyclic N1 groups of the guanine nucleotide base, whereas the hydroxyl of Ser414 and the backbone amide of Lys331 hydrogen bond to the 6-oxo position. The side chains of Lys331 and Pro417 pack against opposite faces of the guanine nucleotide base. The synthetase recognizes neither the N7 position of guanine nucleotides nor the ribose group. Electron density for the guanine-5'-(beta,gamma-imido) triphosphate complex is consistent with a mixture of the triphosphate nucleoside and its hydrolyzed diphosphate nucleoside bound to the active site. The base, ribose, and alpha-phosphate positions overlap, but the beta-phosphates occupy different binding sites. The binding of guanosine-5'-(beta,gamma-methylene)triphosphate to the active site is comparable with that of guanosine-5'-(beta, gamma-imido)triphosphate. No electron density, however, for the corresponding diphosphate nucleoside is observed. In addition, electron density for bound Mg2+ is absent in these nucleotide complexes. The guanine nucleotide complexes of the synthetase are compared with complexes of other GTP-binding proteins and to a preliminary structure of the complex of GDP, IMP, Mg2+, and succinate with the synthetase. The enzyme, under conditions reported here, does not undergo a conformational change in response to the binding of guanine nucleotides, and minimally IMP and/or Mg2+ must be present in order to facilitate the complete recognition of the guanine nucleotide by the synthetase.     

Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli

Tryptophanyl-tRNA synthetase of Escherichia coli has 1.8 binding sites for L-tryptophan with Kdiss of 12 x 10(-5) M as shown by equilibrium dialysis. The results are in accord with the known structure of the enzyme, and alpha2 dimer of 74,000 molecular weight, and with 2 binding sites for tryptophanyl-ATP ester. Ordinary sucrose density gradient centrifugation reveals a complex composed of one tRNATrp bound per enzyme dimer. When tRNATrp is mixed throughout the gradient at concentrations from 5.4 x 10(-6) M to 2.0 x 10(-5) M, a new peak appears in the position expected for a complex with two tRNATrp molecules bound per enzyme dimer. Sedimentation through gradients lacking tRNATrp favors dissociation of the 1:2 complex but not the 1:1 complex. The data indicate 2 binding sites for tRNATrp on tryptophanyl-tRNA synthetase.     

In vitro studies on ppGpp synthetase I from polyamine-starved and unstarved Escherichia coli

We have studied the in vitro formation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) using a partially purified ppGpp synthetase I (PSI) from Escherichia coli BGA8, a polyamine auxotrophic strain. A comparison of the enzyme obtained from polyamine-supplemented or deprived bacteria showed similar requirements for the reaction, Mg+2 optimum levels and sparing effect of spermidine. No differences in the inhibitory effects of tetracycline, puromycin and fusidic acid were detected either. However, a modified subcellular distribution, as well as a larger specific activity and a larger stimulation by streptomycin was observed when PSI was prepared from polyamine-depleted bacteria. The role of ribosome assembly and subunit distribution on the altered properties of the enzyme are discussed.     

Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Design, synthesis and evaluation of transition-state analogue inhibitors of Escherichia coli gamma-glutamylcysteine synthetase

Phosphinic acid-, sulfoximine- and sulfone-based transition-state analogues were synthesized and evaluated as inhibitors of Escherichia coli gamma-glutamylcysteine synthetase. These compounds have a carboxyl function at the beta-carbon to the tetrahedral central hetero atom so as to mimic the carboxyl group of the attacking cysteine in the transition state. The phosphinic acid- and the sulfoximine-based compounds were found to be potent ATP-dependent inactivators, both showing a slow-binding kinetics with overall affinities and second-order inactivation rates of one to two orders of magnitude greater than those of L-buthionine (SR)-sulfoximine (L-BSO). The sulfone was a simple reversible inhibitor without causing ATP-dependent enzyme inactivation, but its affinity toward the enzyme was still five times greater than that of L-BSO, indicating that the beta-carboxyl function plays a key role in the recognition of the inhibitors by the enzyme. The sulfoximine with (S)-beta-carbon to the sulfur was synthesized stereoselectively, and the two diastereomers with respect to the chiral sulfur atom were separated as a cyclic sulfoximine derivative. The sulfoximine with R-configuration around the sulfur served as an extremely powerful ATP-dependent inactivator with an overall inhibition constant of 39 nM and an inactivation rate of 6750 M-1 s-1, which correspond to 1260-fold higher affinity and almost 1400-fold greater inactivation rate as compared with L-BSO. The sulfoximine with (S)-sulfur was a simple reversible inhibitor with an inhibition potency comparable to that of the sulfone. The synthesis and inhibition profile of the N-phosphoryl sulfoximine is also described.