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PubMed1980

Properties and organ distribution of ATP citrate (pro-3S)-lyase.

1. The lipogenic enzyme ATP citrate lyase (ATP:citrate oxaloacetate-lyase (pro-3S-CH2COO-acetyl-CoA; ATP-dephosphorylating), EC 4.1.3.8) is partially purified from human liver by ammonium sulfate fractionation and anionexchange chromatography. 2. Km values for the substrates are 1.1 x 10(-5) 1.3 x 10(-3), and 1.2 x 10(-4) M for CoASH, ATP and citrate, respectively. The hypolipidemic drug L(-)-hydroxycitrate is a competitive inhibitor with respect to citrate (Ki = 3 x 10(-4) M). 3. Specific activities measured in liver, adipose tissue and intestinal mucosa (autopsic and biopsic material) are in the range of 1 mU/mg protein suggesting that the citrate pathway does not significantly contribute to human lipogenesis. No stimulation is found after a 3-day carbohydrate-rich diet. 4. Specific activities of other key-enzymes of the acetyl-CoA production from carbohydrates (pyruvate dehydrogenase, cytosolic acetyl-CoA synthetase) are of the same low magnitude.

ATP Citrate (pro-S)-Lyase↗
PubMed2006

Active-site assembly in glutaminyl-tRNA synthetase by tRNA-mediated induced fit.

Structure-based mutational analysis was employed to probe an unusual intramolecular interaction between partially buried glutamate residues adjacent to the active site of Escherichia coli glutaminyl-tRNA synthetase (GlnRS). The crystal structures of unliganded GlnRS and the GlnRS-tRNA(Gln) complex reveal that the Glu34 and Glu73 side chain carboxylates contact each other only in the tRNA-bound state and that the interaction is formed via mutual induced-fit transitions that occur en route to the ground-state Michaelis complex. Steady-state and transient kinetic analysis of mutant enzymes suggest that the formation of this intermolecular contact is a key event that facilitates the proper formation of the active site. Mutants at both positions destabilize the binding of the substrate glutamine at the opposite side of the active-site cleft, whereas Glu73 appears to play an additional important role by promoting the correct binding of the 3'-acceptor end of tRNA adjacent to both ATP and glutamine. The data suggest the existence of multiple structural pathways by which the binding of tRNA propagates conformational transitions leading to the proper formation of the glutamine binding site. The single-turnover kinetic analysis also establishes that the Glu34 carboxylate does not play a direct enzymatic role as a catalytic base to help deprotonate the tRNA-A76 nucleophilic 2'-hydroxyl group. The elimination of this previously proposed mechanism, together with recent chemical modification experiments in the histidyl-tRNA synthetase system, emphasizes that substrate-assisted catalysis by the phosphate of the aminoacyl adenylate may be a common means by which all tRNA synthetases facilitate the aminoacyl transfer step of the reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Expression of human aspartyl-tRNA synthetase in Escherichia coli. Functional analysis of the N-terminal putative amphiphilic helix.

Mammalian aspartyl-tRNA synthetase occurs in the multienzyme complex of aminoacyl-tRNA synthetases, while bacterial and yeast aspartyl-tRNA synthetases exist as free soluble enzymes. Cloning and sequencing of mammalian aspartyl-tRNA synthetase revealed a newly evolved N-terminal 32-amino-acid sequence, which contains a putative amphiphilic helix (Jacobo-Molina, A., Peterson, R., and Yang, D. C. H. (1989) J. Biol. Chem. 264, 16608-16612). Human aspartyl-tRNA synthetase (hDRS) and an N-terminal 32-residue truncated form of human aspartyl-tRNA synthetase (hDRS delta 32) were expressed in Escherichia coli under the control of the inducible tac promoter as glutathione-S-transferase (GST) fusion proteins linked through a thrombin cleavage site. The GST-hDRS fusion protein and the GST-hDRS delta 32 were purified by affinity chromatography on glutathione-agarose and were fully active in aspartylation of mammalian tRNA. After cleavage of GST from the fusion proteins by thrombin, hDRS and hDRS delta 32 were purified by affinity chromatography on tRNA-Sepharose. Both hDRS and hDRS delta 32 were present as a mixture of monomeric and dimeric forms. GST-hDRS formed high molecular weight aggregates while GST-hDRS delta 32 was a dimeric protein. Both hDRS and hDRS delta 32 bound to hydrophobic interaction gels such as aminohexyl-agarose. In the absence of propylene glycol, hDRS bound to amino-hexyl-agarose weaker than hDRS delta 32, but, in the presence of 50% propylene glycol, hDRS bound tighter than hDRS delta 32. Both hDRS and hDRS delta 32 were fully active in aspartylation of mammalian tRNA and ATP-PPi exchange. In comparison to the N-terminal truncated form, the full-length enzyme showed greater thermal stability and ATP-PPi exchange activity but lower aminoacylation activity. The catalytic constant of hDRS delta 32 for aminoacylation of tRNA was 2-fold higher than that of hDRS. The Michaelis-Menten constants for aspartic acid and tRNAAsp were 302 microM and 13 nM for hDRS, and 29 microM and 130 nM for hDRS delta 32, respectively. These results suggest that the newly evolved N-terminal peptide in hDRS may modulate the enzymatic activity, the stability, and the chromatographic behavior of hDRS. The structure and function of the N-terminal peptide in aspartyl-tRNA synthetase and in the synthetase complex will be discussed.

Adenosine Triphosphate↗
PubMed2002

Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex.

It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.

Animals↗
PubMed1981

Catalytic mechanism of valyl-tRNA synthetase from baker's yeast. Reaction pathway and rate-determining step in the aminoacylation of tRNAVal.

The catalytic mechanism of valyl-tRNA synthetase from baker's yeast has been investigated by pre-steady-state and steady-state kinetic measurements and end product dissociation studies. The pre-steady-state kinetics show a lag period during the early time when the reaction is started with free enzyme. The preincubation of the synthetase with tRNAVal and/or valine or preformation of Val approximately AMP leads to a progressive suppression of the lag. This lag probably reflects conformational transitions of the enzyme-substrate complex necessary for the transfer. At low pH or at a low ionic strength, the tRNAVal charging occurs much faster at the pre steady state than at the steady state. We show that after the fast transfer of valine from adenylate to tRNAVal, followed by the fast dissociation of AMP and PPi, a new adenylate is synthesized which promotes the dissociation of the nascent Val-tRNAVal. This dissociation occurs in a multistep process. First ATP and magnesium promote the ejection of the valine moiety of Val-tRNAVal from the adenylate site. A new adenylate is then synthesized which promotes, in the presence of magnesium, several state changes of the end product complex. A complex is finally generated in which the enzyme-bound Val-tRNAVal is able to exchange rapidly with a tRNAVal molecule. The free tRNAVal plays an active role in this exchange. Depending upon the experimental conditions, one of these steps can determine the steady-state rate of tRNAVal charging. The dissociations of enzyme-bound uncharged tRNAVal or aa-tRNAs substituted on the amino acid or on the tRNA parts by noncognate parts as well as the effect of the replacement of the adenylates by wrong adenylates have been investigated. It is shown that the valine and the tRNA moieties of Val-tRNAVal and the valine moiety of the adenylate are involved in this mechanism of dissociation. Finally, the rate-determining step of the reversal of tRNAVal charging at the steady-state has been investigated. It is shown that this step is the dissociation of the deacylated tRNAVal from enzyme.

Acylation↗
PubMed2004

Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa. In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ. We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography. PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined. The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain. The domains are intricately linked by interdomain interactions and peptide connectors. The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members. A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site. These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site. A glutamylthioester intermediate was found in the glutaminase domain at Cys1135. The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.

Adenosine Diphosphate↗
PubMed2000

Heat shock protein 90 mediates protein-protein interactions between human aminoacyl-tRNA synthetases.

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

Adenosine Triphosphate↗
PubMed2003

Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli.

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.

Adenosine Triphosphate↗
PubMed1983

Bovine lens gamma-glutamylcysteine synthetase. Inhibition by glutathione and adenine nucleotides.

Steady-state kinetic analysis shows that glutathione binds reversibly to both Mg . enzyme and Mg . enzyme . L-glutamate forms of gamma-glutamylcysteine synthetase to form inactive complexes. The Ki values for binding to these two species of enzyme are 4 mM and 0.4 mM, respectively; those for S-methyl glutathione are 16 mM and 0.5 mM, respectively. These data suggest that glutathione is an important feedback inhibitor and contributes to the regulation of glutathione synthesis by modulating the synthesis rate of the precursor dipeptide. Adenosine 5'-diphosphate (5'ADP) is also an inhibitor and competes with both ATP and L-beta-chloroalanine for Mg . enzyme . L-glutamate and Mg . enzyme . L-glutamylphosphate, respectively. Under physiological conditions in the lens, 5' ADP competes effectively with L-cysteine for Mg . enzyme . L-glutamylphosphate, owing to the low concentration of L-cysteine, and less effectively with ATP for Mg . enzyme . L-glutamate, because of a high concentration of ATP.

Adenine Nucleotides↗
PubMed1996

Novel isolation method and structural stability of a eukaryotic chaperonin: the TCP-1 ring complex from rabbit reticulocytes.

In the course of removing a contaminant from preparations of aminoacyl-tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin-agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino-terminal sequencing, and ATP-dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the "characteristic" ring and four-stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate-containing views are observed. The particle structure is strikingly resistant to physical forces (long-term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KCl or LiCl), and pH changes (9-6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KCl or deoxycholate reduces TRiC folding activity.

Amino Acid Sequence↗
PubMed1984

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase.

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.

Amino Acid Isomerases↗
PubMed1986

Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes.

The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.

Brevibacterium↗
PubMed1997

Engineering of glycerol-stimulated insulin secretion in islet beta cells. Differential metabolic fates of glucose and glycerol provide insight into mechanisms of stimulus-secretion coupling.

Insulin secretion from beta cells in the islets of Langerhans can be stimulated by a number of metabolic fuels, including glucose and glyceraldehyde, and is thought to be mediated by metabolism of the secretagogues and an attendant increase in the ATP:ADP ratio. Curiously, glycerol fails to stimulate insulin secretion, even though it has been reported that islets contain abundant glycerol kinase activity and oxidize glycerol efficiently. We have reinvestigated this point and find that rat islets and the well differentiated insulinoma cell line INS-1 contain negligible glycerol kinase activity. A recombinant adenovirus containing the bacterial glycerol kinase gene (AdCMV-GlpK) was constructed and used to express the enzyme in islets and INS-1 cells, resulting in insulin secretion in response to glycerol. In AdCMV-GlpK-treated INS-1 cells a greater proportion of glycerol is converted to lactate and a lesser proportion is oxidized compared with glucose. The two fuels are equally potent as insulin secretagogues, despite the fact that oxidation of glycerol at its maximally effective dose (2-5 mM) occurs at a rate that is similar to the rate of glucose oxidation at its basal, nonstimulatory concentration (3 mM). We also investigated the possibility that glycerol may signal via expansion of the glycerol phosphate pool to allow enhanced fatty acid esterification and formation of complex lipids. Addition of Triacsin-C, an inhibitor of long-chain acyl-CoA synthetase, to AdCMV-GlpK-treated INS-1 cells did not inhibit glycerol-stimulated insulin secretion despite the fact that it blocked glycerol incorporation into cellular lipids. We conclude from these studies that glycerol kinase expression is sufficient to activate glycerol signaling in beta cells, showing that the failure of normal islets to respond to this substrate is due to a lack of this enzyme activity. Further, our studies show that glycerol signaling is not linked to esterification or oxidation of the substrate, but is likely mediated by its metabolism in the glycerol phosphate shuttle and/or the distal portion of the glycolytic pathway, either of which can lead to production of ATP and an increased ATP:ADP ratio.

Adenoviridae↗
PubMed1982

Catalytic cycle of the biosynthetic reaction catalyzed by adenylylated glutamine synthetase from Escherichia coli.

The covalently attached AMP moiety of adenylylated glutamine synthetase from Escherichia coli has been replaced by its fluorescent analog, 2-aza-1,N6-etheno-AMP (aza-epsilon-AMP). The modified glutamine synthetase (aza-epsilon-GS) exhibits divalent cation requirement (Mn2+, rather than Mg2+), pH profile, Vmax, and Km similar to those of naturally adenylylated glutamine synthetase. Whereas naturally adenylylated glutamine synthetase exhibits only negligible fluorescence changes upon the binding of substrates, aza-epsilon-GS exhibits large fluorescence changes. The fluorescence changes have been used by means of a stopped flow technique to reveal the involvement of five fluorometrically distinct intermediates in the catalytic cycle for the biosynthesis of glutamine catalyzed by the adenylylated glutamine synthetase. The mechanism is very similar to that previously established for the unadenylylated enzyme, using intrinsic tryptophan fluorescence. Substrates bind via a rapid equilibrium random mechanism, but the reaction proceeds in a stepwise manner. The formation of an enzyme-bound intermediate (probably gamma-glutamyl phosphate + ADP) from ATP and L-glutamate is the rate-limiting step, with the subsequent reaction of the enzyme-bound intermediate occurring very rapidly. The success in elucidating this complex mechanism is due largely to the vastly different amplitudes of the fluorescence changes at the two excitation maxima (300 nm and 360 nm) of the aza-epsilon-AMP moiety which accompany the formation of the various intermediates.

Adenosine Monophosphate↗
PubMed1988

A mathematical model of the Calvin photosynthesis cycle.

1. A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium steady-state and transient-state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6-phosphate, glucose 1-phosphate, ATP, ADP, and inorganic (ortho)phosphate. 2. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. 3. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady-state cycle activity and rate of starch production. 4. Control analyses are reported which show that most of the non-equilibrium enzymes in the system have a strong regulatory influence on the steady-state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady-state concentration of the substrate is consumes. 5. Under optimal external conditions (0.15-0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbohydrates↗
PubMed1998

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K4 = 1.7 +/- 0.9 microM) approximately 35-fold more tightly than to E (K1 = 50-100 microM), primarily due to a reduction in the Ile dissociation rate constant (k-1 approximately 100-150 s-1, cf. k-4 = 3 +/- 1.5 s-1). Similarly, ATP binds more tightly to E.Ile (K3 = approximately 70 microM) than to E (K2 = approximately 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k+5 = approximately 60 s-1) and the reverse rate constant (k-5 = approximately 150-200 s-1) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K6 = approximately 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (kcat,tr = approximately 0.35 s-1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.

Adenosine Triphosphate↗
PubMed2006

Expression, purification, crystallization and preliminary X-ray diffraction analysis of human phosphoribosyl pyrophosphate synthetase 1 (PRS1).

Phosphoribosyl pyrophosphate synthetase (PRS; EC 2.7.6.1) catalyzes the reaction of ribose-5-phosphate (R5P) with ATP to yield AMP and PRPP (5-phosphoribosyl-1-pyrophosphate), which is necessary for the de novo and salvage pathways of purine-, pyrimidine- and pyridine-nucleotide biosynthesis. PRPP is a metabolite that is required at all times in the cell and is thus central to life. In this study, human PRS1 was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals in complex with Mg2+, inorganic phosphate (P(i)) and ATP were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.6 A resolution. The crystal belongs to space group R3, with unit-cell parameters a = b = 168.846, c = 61.857 angstroms, assuming two molecules in the asymmetric unit and a volume-to-weight ratio of 2.4 angstroms3 Da(-1), which was consistent with the result calculated from the self-rotation function.

Brain↗
PubMed1985

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase.

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.

Amino Acyl-tRNA Synthetases↗