Amino Acid-activating Enzymes Research Articles

Functional Class I and II amino acid-activating enzymes can be coded by opposite strands of the same gene.

VOLUME 290 (2015) PAGES 19710–19725 There were several errors in this article. Manual transcription of sequence data into Figs. 2 and 6 inadvertently introduced errors that were identified and communicated to the corresponding author by a reader. The corrected figures are provided. Additionally, on page 19723, the Class II phylogenetic signature under the “Sense/Antisense 46-mers May Represent a Transition between Uncoded and Genetically Coded Peptides” section should be VTDXXXXR. These errors do not alter either the results or conclusions of the work.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or ∼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Functional Class I and II Amino Acid-activating Enzymes Can Be Coded by Opposite Strands of the Same Gene

Structural and functional organization of the surfactin synthetase multienzyme system.

By gel filtration of a crude extract of Bacillus subtilis ATCC 21332 and OKB 105, the multienzyme system that forms the lipoheptapeptide surfactin was separated into three enzyme fractions, E1, E2, and E3. E1, which appeared near the exclusion limit of the column, activates all amino acid components of surfactin as aminoacyladenylates and thioesters according to the thioester mechanism. In addition, a leucine-activating enzyme (E2) and an acyltransferase (E3) were detected that show molecular masses of approximately 160 and 40 kDa, respectively. The surfactin synthetase multienzyme system was reconstituted by complementation of all three enzyme fractions, yielding high rates of lipopeptide formation. E1 is composed of two multifunctional polypeptides (E1A and E1B) with molecular masses of 460 and 435 kDa, respectively, that can be separated by high-resolution anion-exchange chromatography on Pharmacia Mono Q. E1A binds L-Glu and L-Leu in a molar ratio of 1:2, whereas E1B incorporates L-Val, L-Asp, and L-Leu in a molar ratio of 1:1:1. The hydroxy fatty acid moiety is contributed by the acyltransferase accepting the hydroxy fatty acid coenzyme A thioester as substrate. The transfer of the hydroxy fatty acid to E1A and the formation of the hydroxyacyl-L-glutamate intermediate are the initiation steps in the biosynthesis of surfactin. The amino acid-activating enzyme components E1A, E1B, and E2 have been highly purified and partially characterized.

Biosynthesis of bacillomycin D by Bacillus subtilis Evidence for amino acid-activating enzymes by the use of affinity chromatography

Bacillomycin D is an antifungal lipopeptide produced by B. subtilis. The formation of the peptidyl bonds of bacillomycin D occurs non-ribosomally, as demonstrated by the use of chloramphenicol, an inhibitor of protein biosynthesis. Amino acid-activating enzymes were found in B. subtilis cell lysates purified by affinity chromatography on a gel containing l-Pro, an amino acid of bacillomycin D. Presence of ATP during this purification increases the binding of enzymatic proteins and their activity. An enzyme, with an apparent molecular weight of 230 kDa, catalyzed ATP-PPi exchange reactions, which were mediated by specific amino acids, corresponding to a partial sequence of bacillomycin D.

Biosynthesis of polymyxin by Bacillus polymyxa: I. The status of the biosynthetic multienzyme complex during active antibiotic synthesis and sporulation

The involvement of membrane fractions of Bacillus polymyxa in the early stages of the biosynthesis of the peptide antibiotic polymyxin, was established by (a) incorporation of the precursor amino acid in an acid precipitable form in the absence of protein synthesis, (b) presence of all the component amino acid-activating enzymes, and (c) association of bioassayable polymyxin, in the purified membrane fraction. Polymyxin negative mutants that were also blocked at stage 0 of sporulation were shown to be defective in one or more of their membrane-bound amino acid-activating enzymes. A strong correlation between sporulation and antibiotic production had been indicated by the isolation of these mutants which are revertible simultaneously to Ab + and Spo + traits. During the onset of the rapid phase of the elaboration of polymyxin, a delocalization of one of the membrane-bound enzymes, 2,4-diaminobutyric acid-activating enzyme, to the soluble fraction was observed. Concomitant with this change, the levels of the intracellular protein-bound and free polymyxin was increased in the soluble fraction.

Biosynthesis of polymyxin by Bacillus polymyxa: II. On the nature and interaction of the multienzyme complex with the end product polymyxin

The interaction of polymyxin with the producer organism Bacillus polymyxa has been shown to be at the level of membranes, resulting in an enhancement of the activities of its own biosynthetic enzymes. This enhancement has been shown to be due to the solubilization of the membrane-associated multienzyme complex by polymyxin in a specific manner. The relevance of this physiological feature was also indicated by the appearance of the soluble multienzyme complex activity only in cells, which synthesize maximal amounts of polymyxin. Purification of the polymyxin released multienzyme complex from the membranes and the soluble form of the complex from the stationary phase cells has revealed several similarities between them. Both contain two major fractions of the molecular weights of 300,000 and 170,000, harboring all the polymyxin component amino acid-activating enzymes. Their multisubunit nature was established by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Using mutants blocked in sporulation and/or antibiotic synthesis, it was shown that the interaction of polymyxin with the producer organism was inoperative when antibiotic production was curtailed. This interaction has been suggested as one of the early sporulation-specific phenemenon.

Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.

The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.

Amino acid activation by nucleoli isolated from the novikoff ascites tumor

The mechanism of the incorporation of amino acids into nucleolar protein has been studied with nucleoli isolated from the Novikoff ascites rat tumor. Each of 15 labeled amino acids, added singly, was incorporated without need for added ATP, tRNA, complementary amino acids, or other components of the cytoplasmic proteinsynthesizing system. The activating enzymes corresponding to most of these amino acids were demonstrated by measuring the rate of amino acid hydroxamate formation when the nucleoli were incubated with the amino acid, NH 2OH and ATP. Evidence for the intermediate formation of an aminoacyl tRNA complex was obtained. The isolated nucleoli appear to contain a tightly-integrated protein-synthesizing system which includes amino acid-activating enzymes and tRNAs.

The inhibition of amino acid-activating enzymes and leucine aminopeptidase by chloromethyl ketone analogues of amino acids.

The inhibition of amino acid-activating enzymes and leucine aminopeptidase by chloromethyl ketone analogues of amino acids.

Transfer RNA species in normal and leukemic human lymphoblasts

The results of a study to identify the transfer RNA's for every amino acid in human tissues are reported. The isoaccepting species of the tRNA of human lymphoblasts (normal and leukemic) were fractionated by reverse-phase partition column chromatography. In every case, comparisons were made between the aminoacyl-tRNA profile of normal and leukemic lymphoblasts by co-chromatography using 3H for one label and 14C for another and the homologous amino acid-activating enzyme. Differences between the profiles of normal and leukemic cells were re-evaluated by reversal of the labeled amino acid and by acylation with the heterologous enzyme. The results indicate that (1) there are at least 56 species of tRNA fractionated from both the normal and the leukemic cells; (2) for most cases the profiles of the leukemic cells were very similar if not identical to the profiles of normal cells; (3) small but reproducible differences were found for leucyl-, seryl-, threonyl- and prolyl-tRNA; (4) the most pronounced differences were with tyrosyl-tRNA and glutaminyl-tRNA. The difference in the glutaminyl-tRNA was an extra isoaccepting species found only when normal lymphoblast enzyme preparation was used. Therefore, in this case the difference was due to the enzyme preparation rather than the tRNA itself.

Induced Activation of Amino Acid Activating Enzymes by Amino Acids and †RNA

This chapter discusses that a characteristic of protein synthesis is the absolute specificity with which the correct amino acids are incorporated into each position of the polypeptide chain. This requires at least the same degree of specificity in the ability of amino acid activating enzymes to discriminate both among potential amino acid substrates and among the many species of tRNA that exist within each cell. It reviews that the most completely analyzed enzymes are hydrolytic, and the success in correlating chemical mechanisms with the structures in the cases of lysozyme, ribonuclease, chymotrypsin, and a few others has resulted in the popular acceptance of a general model of enzymes. Enzymes that function in an extracellular environment tend to have characteristics different from those of intracellular catalysts. The amino acid-activating enzymes are a family of catalysts related to a single biological phenomenon, protein synthesis, and to a single formal reaction. The chapter focuses on those properties that may give insight into the mechanism of action of individual activating enzymes, which includes the chemical basis for specific recognition. It is proposed that the combination of an enzyme with a specific substrate results in conformational changes that may result in the formation of a catalytic site, in the binding site for another substrate, or in the approximation of one site to another.

L-α,γ-Diaminobutyrate-activating enzyme from Bacillus polymyxa: Properties and distribution

1. 1. An enzyme which catalyses the l-α,γ-diaminobutyrate-dependent exchange of ATP with PP i was partially purified from sonic extracts of the polymyxin B-producing organism, Bacillus polymyxa 2459. This enzyme could be separated from most other amino acid-activating enzymes and exhibited an apparent K m for l-α,γ-diaminobutyrate of 0.6 mM. 2. 2. Treatment of sonic extracts with streptomycin sulfate led to an alteration in the properties of the activating enzyme. This appeared to be due to the conversion of the enzyme from polydisperse aggregates of average molecular weight 300 000 to units of 100 000 molecular weight. 3. 3. In lysed protoplast preparations, the l-α,γ-diaminobutyrate-activating enzyme was predominantly associated with the particulate fraction, in contrast with other amino acid-activating enzymes. The K m for l-α,γ-diaminobutyrate of the particulate preparation was the same as that of the soluble enzyme. 4. 4. The specific activity of the l-α,γ-diaminobutyrate-activating enzyme rose about 10-fold during vegetative growth, beginning at the time of the first appearance of polymyxin in the growth medium. 5. 5. Mutants of B. polymyxa that had lost the ability to produce polymyxin also lacked the l-α,γ-diaminobutyrate-activating enzyme. On the other hand, other polymyxin-producing organisms, such as B. polymyxa ATCC 10401 and B. circulans ATCC 14040, were found to have significant levels of this enzyme. These observations suggest that the l-α,γ-diaminobutyrate-activating enzyme plays a role in the biosynthesis of polymyxin.

Mechanism of Action of Amino Acid Transfer Ribonucleic Acid Ligases

The first reaction in protein biosynthesis has been presumed to be the interaction of ATP, amino acid, and enzyme (amino acid-activating enzyme, aminoacyl transfer RNA ligase, tRNA synthetase) to form an enzyme-bound aminoacyl adenylic anhydride. Such aminoacyl anhydrides could react forward with hydroxylamine or with tRNA or backward with [32P]-PPi. Experiments, primarily involving inhibitors, cast serious doubt on the existence of such a common intermediate in the enzyme-catalyzed formation of amino acid hydroxamate or [32P]-ATP from amino acid, ATP, and hydroxylamine, or [32P]-PPi, respectively. Studies with several enzymes indicate the probability of a general base-stimulated concerted reaction in which amino acid hydroxamate, AMP, and PPi are formed with no discrete intermediates. The esterification of tRNA resembles hydroxamate formation more than it resembles ATP-PPi exchange, strengthening the argument that the physiologically significant reaction does not involve an enzyme-bound aminoacyl adenylate. When tRNA is esterified in the absence of other bases, it is probable that one of the terminal ribose hydroxyls serves as a general base while the other is being acylated.

Selection of temperature-sensitive activating enzyme mutants in Escherichia coli.

A method based on temperature-conditional resistance to thymineless death has been designed to facilitate the isolation of amino acid-activating enzyme mutants. This method may be modified to obtain a greater or lesser proportion of activating enzyme mutants. In the latter instance, an increased proportion of temperature-sensitive macromolecule mutants of other types is obtained. Additional uses of this procedure are discussed.

EFFECT OF MALNUTRITION ON ACTIVITY OF TWO ENZYMES CONCERNED WITH AMINOACID METABOLISM IN HUMAN LIVER

Aminoacid-activating enzymes and argininosuccinase were measured in liverbiopsy samples from twenty-two malnourished and recovering Jamaican infants. Aminoacid-activating-enzyme levels were increased initially and fell on recovery; argininosuccinase activity was low initially and rose on recovery. It is suggested that these changes are part of the adaptation which takes place in aminoacid metabolism in the malnourished state.

The Arginyl Transfer Ribonucleic Acid Synthetase of Escherichia coli

Abstract The arginyl transfer ribonucleic acid (tRNA) synthetase of Escherichia coli has been purified over 500-fold. Its kinetic properties are similar to those of other amino acid-activating enzymes; it has a pH optimum near 8 and does not react with amino acids that occur in proteins other than arginine, but the analogues homoarginine and canavanine are competitive inhibitors, and canavanine can be esterified with tRNA. The ATP-pyrophosphate exchange reaction is not catalyzed in the presence of arginine unless arginyl-tRNA is also present.

Effect of a Methionine-deficient Diet on Amino Acid Incorporation in Rat Liver Cell-fractions

An investigation was made into the relationship between nutritive value of a protein-rich food and amino acid incorporation into cell-free preparations from rat liver. A low protein efficiency ratio due in part to a methionine deficiency was associated with increased levels of protein labeling. The effect was observed with methionine-35S but not with leucine-14C, and was apparently mediated through alterations occurring predominantly in the pH 5-enzyme fraction. The observations indicated that an increase in the activity of amino acid-activating enzymes may be induced by dietary deficiency of the corresponding amino acids.

Amino Acid Activation in the Liver of Growing Rats Maintained with Normal and with Protein-deficient Diets

The activity of the amino acid-activating enzymes was measured in the liver of growing rats maintained with a normal and with a protein-deficient diet. The determinations were based on the isotope exchange between ATP and PP labeled with 32P. The changes in the DNA content of the cell nucleus were also determined. Studies of changes in DNA content, and hence changes in ploidy during growth, indicated that in normal rats the activity of the amino acid-activating enzymes increased when expressed per average nucleus, whereas it remained constant and unaffected by growth when expressed per unit weight of DNA. On the contrary, in rats maintained with a protein-deficient diet, in which the arrest of growth causes an arrest of ploidy, the activity of the amino acid-activating enzymes increased both when expressed per average nucleus and per unit weight of DNA. We suggest that under these conditions a control mechanism is brought into action by changes in the amount of amino acids in circulation. This mechanism is independent of the increase in the DNA content of cell nucleus, and results, for growing rats maintained with a protein-deficient diet, in a preferential utilization of the amino acids for the synthesis of proteins.

Some studies of the association of amino acid-activating enzymes with isolated microsomes of chick embryo

The association of thirteen amino acid-activating enzymes with microsomal preparations from homogenated chick embryo has been studied by using the amino acid-dependent, adenosine triphosphate-inorganic pyrophosphate-P 32 (ATP-P 32P i 32) exchange assay. It was found that a substantial portion of the total activity of the amino acid-activating enzymes is associated with the microsomal fraction under the conditions employed. Extensive washing of the microsomes releases a portion of the activity of some of the amino acid-activating enzymes tested, but over 90% of the activity of the phenylalanine- and tyrosine-activating enzymes remains associated with the microsomes after four washes. Over 80% of the microsome-bound proline activation activity is destroyed by extensive washing, but if proline is present in the wash medium, over 70% of the proline activation activity remains associated with the microsomal fraction. The effects of treatment of the microsomes with sodium deoxycholate were studied, and this compound was found to completely inactivate most of the amino acid-activating enzymes. The lysine-, arginine-, and threonine-activating enzymes are largely unaffected by sodium deoxycholate treatment, and most of the activity of these enzymes is released from the microsomes into the soluble fraction.