Cysteinyl-tRNA Synthetase from Phaseolus aureus: Purification and Properties

l-Cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus was purified approximately 300-fold and was free of contaminating aminoacyl-tRNA synthetases. Optimum assay conditions were determined and substrate specificity and inhibitor properties were investigated using the ATP-PPi exchange reaction. The Km values for l-cysteine, ATP, and PPi were 6.20 x 10(-5)m, 1.15 x 10(-3)m, and 1 x 10(-3)m, respectively. Both l-selenocysteine (Km = 5 x 10(-5)m) and alpha-l-aminobutyric acid (Km = 1 x 10(-2)m) acted as alternative substrates of the purified cysteinyl-tRNA synthetase. The enzyme was sensitive to sulfhydryl group reagents; it was inhibited by sulfide, 0-acetylserine, and reduced glutathione.     

Dissimilation of methionine by a demethiolase of Aspergillus species

Enzyme preparations obtained from the mycelium of Aspergillus species broke down methionine by co-dissimilation. The deaminase and demethiolase activities of crude extracts were increased 100-fold by precipitation with (NH(4))(2)SO(4) and column chromatography on diethylaminoethyl cellulose. The enzyme acted on d-methionine but not on l-methionine. The enzyme was labile: it was inactivated by oxygen and ascorbic acid but ethylenediaminetetraacetic acid and mercaptoethanol preserved its activity. Enzyme activity decreased even at 4 and -30 C and was lost rapidly above 45 C. It was most rapid at 35 C and at pH 8.0 to 9.0. For the following reasons, it was concluded that deamination and demethiolation of methionine were effected by the same enzyme: both activities increased equally at each stage of purification; ammonia, methanethiol, and alpha-keto butyric acid were formed in amounts equivalent to the amount of methionine dissimilated; the K(m) and optimal pH for formation of both keto acid and methanethiol were the same; both activities remained in the same fractions that were separated by electrophoresis and the activities were equivalent. The purified enzyme demethiolated alpha-keto methionine and alpha-hydroxy methionine and split the sulfur linkage of ethionine but did not cleave cystathionine. Few amino acids were deaminated. The enzyme was sensitive to some carbonyl and sulfhydryl reagents and was relatively insensitive to heavy metals other than Hg(++). The K(m) was 1.3 x 10(-3) to 1.5 x 10(-3)m at pH 7.0. No requirement for cofactors was noted, and attempts to dissociate the enzyme, including dialysis with hydroxylamine, were unsuccessful.     

Purification and properties of galactokinase from pig liver

1. Galactokinase has been purified from the liver of young pigs by high-speed centrifugation, chromatography on Sephadex G-100 and DEAE-cellulose, and ammonium sulphate fractionation. 2. The enzyme preparation has a specific activity of 10-18mumoles of galactose phosphorylated/mg. of protein/min. at 37 degrees and has been purified 400-fold from the liver supernatant. 3. Purified liver galactokinase has Michaelis constants of 1x10(-4)-3x10(-4)m for galactose and 2x10(-4)m for ATP-Mg(2+), and the enzyme reaction produces equimolar amounts of galactose 1-phosphate and ADP. 4. Galactokinase phosphorylates 2-deoxygalactose and galactosamine in addition to galactose, has a pH optimum of 7.8, a Q(10) of 2, and is stimulated by cysteine and other thiols. 5. With the exception of substrate specificity, the properties of liver galactokinase are similar to galactokinase purified from yeast and Escherichia coli.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Leucyl-transfer ribonucleic acid synthetase from a wild-type and temperature-sensitive mutant of Salmonella typhimurium

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following K(m) values: leucine, 1.1 x 10(-5)m; adenosine triphosphate, 6.5 x 10(-4)m; tRNA(I) (Leu), 4.1 x 10(-8)m; tRNA(II) (Leu), 4.3 x 10(-8)m; tRNA(III) (Leu), 5.3 x 10(-8)m; and tRNA(IV) (Leu), 2.9 x 10(-8)m. The tRNA(Leu) fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min(-1). Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.     

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold. Two N-[ethyl-2-3H]maleimide-labeled tryptic peptides were purified from the modified enzyme by reverse phase high performance liquid chromatography. The modified residues were identified as cysteine 90 and cysteine 240 by comparison of the amino acid compositions of these peptides with the protein sequence. These are the first residues to be implicated in the activity and/or structure of the enzyme. N-Ethylmaleimide-modified S-adenosylmethionine synthetase exists mainly as a dimer in conditions where the native enzyme is a tetramer. Accumulation of the dimer parallels the loss of the enzyme activity. When an enzyme sample was partially inactivated, separation of tetrameric and dimeric enzyme forms by gel filtration revealed that the residual enzyme activity was solely present in the tetramer and N-[ethyl-2-3H] maleimide was present predominantly in the dimer. Gel filtration studies of the tetramer-dimer equilibrium for the native enzyme indicated that the dissociation constant between the tetramer and dimers is less than 6 x 10(-11) M. Similar studies for the N-ethylmaleimide-modified protein indicated that the dissociation constant of the tetramer is approximately 4 x 10(-4) M. Upon modification the strength of dimer-dimer interactions is diminished by at least 9 kcal/mol.     

Genetic and regulatory aspects of methionine biosynthesis in Saccharomyces cerevisiae

Methionine biosynthesis and regulation of four enzymatic steps involved in this pathway were studied in Saccharomyces cerevisiae, in relation to genes concerned with resistance to ethionine (eth(1) and eth(2)). Data presented in this paper and others favor a scheme which excludes cystathionine as an obligatory intermediate. Kinetic data are presented for homocysteine synthetase [K(m)(O-acetyl-l-homoserine) = 7 x 10(-3)m; K(i) (l-methionine) = 1.9 x 10(-3)m]. Enzymes catalyzing steps 3, 4, 5, and 9 were repressible by methionine. Enzyme 4 (homoserine-O-transacetylase) and enzyme 9 (homocysteine synthetase) were simultaneously derepressed in strains carrying the mutant allele eth(2) (r). Studies on diploid strains confirmed the dominance of the eth(2) (s) allele over eth(2) (r). Regulation of enzyme 3 (homoserine dehydrogenase) and enzyme 5 (adenosine triphosphate sulfurylase) is not modified by the allele eth(2) (r). The other gene eth(1) did not appear to participate in regulation of these four steps. Gene enzyme relationship was determined for three of the four steps studied (steps 3, 4, and 9). The structural genes concerned with the steps which are under the control of eth(2) (met(8): enzyme 9 and met(a): enzyme 4) segregate independently, and are unlinked to eth(2). These results are compatible with the idea that the gene eth(2) is responsible for the synthesis of a pleiotropic methionine repressor and suggest the existence of at least two different methionine repressors in S. cerevisiae. Implications of these findings in general regulatory mechanisms have been discussed.     

N2-guanine specific transfer RNA methyltransferase I from rat liver and leukemic rat spleen

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N(2)-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m(2)G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA(Arg), tRNA(Phe), and tRNA(Val) yielded significantly more m(2)G than the bulk tRNA. The K(m) for tRNA(Arg) in the methylation reaction with enzymes from either tissue was 7.8 x 10(-7) M as compared to the value 1 x 10(-5) M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA(Arg), tRNA(Phe), and tRNA(Val), A-m(2)G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5'-end contained in a sequence (s(4))U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid. We have purified CMP-NeuAc synthetase from an Escherichia coli O18:K1 cytoplasmic fraction to apparent homogeneity by ion exchange chromatography and affinity chromatography on CDP-ethanolamine linked to agarose. The enzyme has a specific activity of 2.1 mumol/mg/min and migrates as a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis. The enzyme has a requirement for Mg2+ or Mn2+ and exhibits optimal activity between pH 9.0 and 10. The apparent Michaelis constants for the CTP and NeuAc are 0.31 and 4 mM, respectively. The CTP analogues 5-mercuri-CTP and CTP-2',3'-dialdehyde are inhibitors. The purified CMP-N-acetylneuraminic acid synthetase has a molecular weight of approximately 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding CMP-N-acetylneuraminic acid synthetase is located on a 3.3-kilobase HindIII fragment. The purified enzyme appears to be identical to the 50,000 Mr polypeptide encoded by this gene based on insertion mutations that result in the loss of detectable enzymatic activity. The amino-terminal sequence of the purified protein was used to locate the start codon for the CMP-NeuAc synthetase gene. Both the enzyme and the 50,000 Mr polypeptide have the same NH2-terminal amino acid sequence. Antibodies prepared to a peptide derived from the NH2-terminal amino acid sequence bind to purified CMP-NeuAc synthetase.     

Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex

Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.     

Properties of D-arabinose isomerase purified from two strains of Escherichia coli

d-Arabinose isomerase (EC 5.3.1.3) has been isolated from l-fucose-induced cultures of Escherichia coli K-12 and d-arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s(20,w) was 14.5 x 10(-13) sec for the E. coli K-12 enzyme and 14.3 x 10(-13) sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 +/- 0.06 x 10(5) for the E. coli K-12 enzyme and 3.42 +/- 0.04 x 10(5) for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l-fucose or d-arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the K(m) was 2.8 x 10(-1)m for d-arabinose and 4.5 x 10(-2)m for l-fucose; with the E. coli B/r enzyme, the K(m) was 1.7 x 10(-1)m for d-arabinose and 4.2 x 10(-2)m for l-fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l-arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn(2+) or Co(2+) ions, but were strongly inhibited by the presence of Cd(2+) ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at pH 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 +/- 0.2 x 10(4) for the enzyme from E. coli K-12 and 8.46 +/- 0.1 x 10(4) for the enzyme from E. coli B/r. On the basis of biophysical studies, both isomerases appear to be oligomeric proteins consisting of four identical subunits.     

Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase

We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product. High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis. The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E. coli. Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter. Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E. coli strain TG2. Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography. Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested. The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E. coli enzyme. E. coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml. At the same concentration of pseudomonic acid A, E. coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1%. The ability of yeast IRS to rescue E. coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E. coli tRNA(ile).     

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.     

Differential inhibition of host and viral thymidylate synthetases by folylpolyglutamates.

The ability of folate analogues to inhibit host and viral thymidylate synthetases was measured using the corresponding Escherichia coli and T2-phage-induced enzymes. In the absence of Mg2+, 6 x 10(-7) M pteroylhexaglutamate inhibited the T2-phage-induced synthetase by 50%, but at least 100-fold greater levels of this compound were necessary to inhibit the E. coli synthetase by this amount. At 2.5 x 10(-6) M pteroylhexaglutamate, at least 80% inhibition of the T2-phage synthetase could be obtained with little or no inhibition of the E. coli enzyme. The pteroylmonoglutamate was about 2 orders of magnitude less inhibitory towards the T2-phage enzyme than the pteroyltri- to -heptaglutamates. However, upon addition of Mg2+ to the assay mixture, the inhibition produced by pteroylhexaglutamate was essentially reversed, with the E. coli synthetase now increasingly inhibited by this compound and the T2-synthetase only minimally impaired. Methotrexate and N10-formyl-2-amino-4-hydroxyquinazoline, although inhibitory to both enzymes in the presence or absence of Mg2+, did not show this differential selectivity. These results suggest that certain folate analogues may be useful in distinguishing between a host and an infecting organism's thymidylate synthetase and could thus provide an additional means of screening for potential chemotherapeutic agents.     

Purification and characterization of specific 3-deoxy-D-manno-octulosonate 8-phosphate phosphatase from Escherichia coli B

A phosphatase specific for the hydrolysis of 3-deoxy-d-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated three- to fourfold by the addition of 1.0 mM Co(+) or Mg(2+) and to a lesser extent by 1.0 mM Ba(2+), Zn(2+), and Mn(2+). The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu(2+), Ca(2+), Cd(2+), Hg(2+), and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 +/- 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent K(m) for KDO-8-phosphate was determined to be 5.8 +/- 0.9 x 10(-5) M in the presence of 1.0 mM Co(2+), 9.1 +/- 1 x 10(-5) M in the presence of 1.0 mM Mg(2+), and 1.0 +/- 0.2 x 10(-4) M in the absence of added Co(2+) or Mg(2+).     

Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8

Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid. The thermostable enzyme thus produced was extracted from the E. coli cells, purified, and crystallized. The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x 10(4). The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M and that for NAD 6.3 x 10(-4) M. The optimum pH at 75 degrees C in the presence of 1.2 M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl. The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein. Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed.     

Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry.

Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of [32P]PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation. Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114-150], [270-362], and [402-465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.     

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.     

Malonyl coenzyme A synthetase. Purification and properties

Malonyl coenzyme A synthetase (EC 6.2.1.14) was induced in Pseudomonas fluorescens grown on malonate as a sole carbon source. This enzyme was purified, for the first time, over 30-fold by the combination of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange chromatography, and hydroxylapatite chromatography. The purified enzyme, which had a specific activity of about 0.512 mumol/min/mg, appeared to be electrophoretically homogeneous. The molecular size of the enzyme was determined to be 98,000 Da which is composed of two 49,000-Da subunits. The optimum pH for the enzyme was 7.5. Malonyl coenzyme A synthetase requires ATP, CoA, and Mg2+ for the full enzyme activity. With succinate or acetate, the synthetic rate of CoA derivative was 40% of that observed with malonate. The malonyl coenzyme A synthetase showed typical Michaelis-Menten kinetics for the substrate, malonate, ATP, and coenzyme A, from which the Km values were calculated to be 3.8 X 10(-4) M, 2 X 10(-3) M, and 10(-4) M and Vmax values to be 0.117 mumol/min/mg, 0.111 mumol/min/mg, and 0.142 mumol/min/mg, respectively. The purified malonyl coenzyme A synthetase was immunogenic in the rabbit and Ouchterlony double diffusion analysis revealed a single precipitant line with the enzyme. The antiserum inhibited the enzyme activity and the extent of inhibition was dependent on the amount of the serum added.     

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.