Chloride affects the interaction between tyrosyl-tRNA synthetase and tRNA.

The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2+, chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration (< 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened the PPi inhibition, the Ki(app)(PPi) values at 1 mM free Mg2+ were 140, 120, and 56 microM at 0, 50 and 150 mM KCl, respectively. Chloride weakened the AMP inhibition, the corresponding values for Ki(app)(AMP) were 0.35, 0.5, and 0.9 mM. The value of Km(app)(tRNA(Tyr)) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Best-fit analyses of the PPi inhibition, AMP inhibition and Km(app)(tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2+ to the tRNA before the transfer reaction and a weakened binding of Mg2+ to the Tyr-tRNA-enzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2+ is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2+ from the aa-tRNA-enzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli.

Differences in the magnesium dependences of the class I and class II aminoacyl-tRNA synthetases from Escherichia coli.

The magnesium dependences of the ATP/PPi exchange and tRNA aminoacylation of reactions were measured for six aminoacyl-tRNA synthetases (isoleucyl-, tyrosyl- and arginyl-tRNA synthetases from class I, and histidyl-, lysyl- and phenylalanyl-tRNA synthetases from class II). The measured values were subjected to best-fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg2+ and to optimize the sets of the kinetic constants. The following four dependences were observed: the class II synthetases require three Mg2+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mg2+ (in MgATP); in class II synthetases both MgPPi and Mg2PPi participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl-tRNA synthetase from class I also shows a better fit if also Mg2PPi reacts, but in the isoleucyl- and tyrosyl-tRNA synthetases only MgPPi but not Mg2PPi is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA-bound Mg2+ and spermidine, independent of the enzyme class. 1-4 Mg2+ or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg2+ from the product aminoacyl-tRNA essentially enhances the subsequent dissociation of the aminoacyl-tRNA from the enzyme. The binding of ATP to the E. aminoacyl-tRNA complex also speeds up the dissociation of the aminoacyl-tRNA from most of these enzymes.

Analysis of the isoleucyl-tRNA synthetase reaction by total rate equations. Magnesium and spermidine in the tRNA kinetics.

Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work [Airas, R.K. (1990) Eur. J. Biochem. 192, 401-409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg(2+)-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.

Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl-tRNA synthetase from Escherichia coli.

A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E.aa-AMP possible. If the E.Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E.Ile-AMP with E.Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E.Ile-AMP and the corrected (*E)tRNA(Ile-AMP). If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.

On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations.

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.

Branched-chain-amino-acid aminotransferase assay using radioisotopes.

A method for the assay of the activity of branched-chain-amino-acid aminotransferase from Escherichia coli has been developed. Radioactive isoleucine is used, the radioactive oxoacid formed in the enzymic reaction is converted to its p-nitrophenylhydrazone, and the hydrazone is extracted into toluene based scintillation liquid. The small reaction tubes containing the toluene layer and the reaction mixture as a water layer are placed into liquid scintillation vials and counted for radioactivity. The radioactive amino acid remaining in the water layer causes only a rather low background.

On the non-linear Eadie plots of the tRNA kinetics and non-linear Dixon plots of the PPi inhibition kinetics of the aminoacyl-tRNA synthetases. An analysis of the aminoacylation of tRNA in a model reaction.

A model of the aminoacyl-tRNA synthetase reaction was analyzed by deriving a rate equation, and by calculating the aminoacylation rates at various values of the rate and equilibrium constants. The model specially contained the possibilities that (1) the activation of the amino acid occurs either with bound or non-bound tRNA, and that (2) the transfer of the aminoacyl moiety from the aminoacyl adenylate to tRNA occurs either with bound or non-bound PPi. The analysis showed that the Eadie plots (tRNA as the variable substrate) are straight lines only if the rates of the activation reactions with bound and non-bound tRNA are equal. Otherwise the Eadie plots can be either curved upwards or downwards. The Dixon plots of the PPi inhibition are straight lines only if PPi must be dissociated from the enzyme before the transfer reaction. The conditions under which the Kiapp values are much lower than the dissociation constants for PPi are met if the transfer reaction is relatively slow and the reverse reaction of the activation (pyrophosphorolysis) is fast, and if the tRNA concentration is low.

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli.

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.

Pantothenases from pseudomonads produce either pantoyl lactone or pantoic acid.

A pseudomonad was separated having a pantothenase that produced beta-alanine and pantoic acid. The pantothenase from an old strain, Pseudomonas fluorescens UK-1, was shown to produce beta-alanine and pantoyl lactone. The pantoic acid-producing pantothenase was characterized and compared with the pantothenase from the strain UK-1. The Mr was 240,000; it apparently consists of four subunits. The Km value for pantothenate is 3 mM. The enzymic activity is affected by an ionizable group of pK 8.4, the enzyme is active at higher pH, and V but not Km is affected by pH. This pantothenase is not inhibited by di-isopropyl phosphorofluoridate or phenylmethanesulphonyl fluoride, unlike the enzyme from the strain UK-1. Both pantothenases are inhibited by m-aminophenylboronic acid, oxalate, oxaloacetate and Cl- ions. The pantoic acid-producing pantothenase is inhibited also by SO4(2-) ions. The strong inhibitions by many compounds make this pantothenase unsuitable for the assay of pantothenic acid.

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa.

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.

Biochemical comparison of the Neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. Detailed kinetic comparison of the leucyl-tRNA synthetases.

The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.

Pantothenase-based assay of pantothenic acid.

Pantothenic acid from various sources can be measured in concentrations from about 10 microM to 20 mM by using a specific enzyme, pantothenase. The method is based on the transfer of [14C]beta-alanine to the pantothenate via an acyl-enzyme intermediate (a beta-alanine-exchange reaction): (formula; see text) Although the reaction is inhibited by some compounds, the method can be used for the assay of pantothenate in many biological samples if an internal pantothenate standard is used.

Kinetic study on the reaction mechanism of pantothenase: existence of an acyl-enzyme intermediate and role of general acid catalysis.

A kinetic study was performed on the reaction mechanism of pantothenase (EC 3.5.1.22) catalyzed hydrolysis of the pantothenic acid. A nonlinear progress curve is derived if the reaction occurs at low buffer concentrations. The nonlinearity is due to partial reversibility of the reaction; an acylenzyme (pantoyl-enzyme) is formed during the reaction, and beta-alanine, the other end product, is able to react with the acyl-enzyme and return back to pantothenate. The dependence of the beta-alanine return reaction on buffer concentration and on pH suggests a general acid catalysis during the reaction. A reaction mechanism is suggested, in which the -NH3+ form of beta-alanine participates in the return reaction, and the deacylation of the acyl-enzyme is acid catalyzed.

On the partial reactivation of inactivated pantothenase from Pseudomonas fluorescens.

Partial reactivation of inactivated pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) from Pseudomonas fluorescens was studied. After partial inactivation during storing, pantothenase activity is increased by 10-40% when incubated with, for instance, oxalate, oxaloacetate or pyruvate. Reactivation proceedes slowly; with oxaloacetate the stable level of enzyme activity is attained in 20-30 min. The same compounds also cause reactivation of thermally inactivated pantothenase when partial inactivation has occurred at 28-37 degrees C. The amount of the reactivating enzyme form is relatively greater the lower the temperature during inactivation, but it never exceeds 20% of the original amount of active enzyme. Also another, unstable form of pantothenase is formed in thermal inactivation. This form becomes inactivated in a few minutes after the heat treatment, at pH 6-8 and at temperatures between 0 and 10 degrees C. Reactivation causes special problems in enzyme kinetic measurements; for instance, curvature is found in the lines of Ki determination by the Dixon plot.

Determination of the binding constant of ligand to protein by thermal inactivation tehnique. Effect of two binding sites.

Theoretical considerations on the determining the binding constants (eta) of ligands to proteins were carried out. Whereas for a one-subunit protein the relationship between thermal inactivation rates and ligand concentration there is a simple linear function, for a protein with two subunits, a second-order relationship is derived. If the theory for one-subunit proteins is applied to multi-subunit proteins, the derived values of eta tend to be lower than the real binding constants. A method of determining the ligand binding constant for a two-subunit protein is described.

Thermal inactivation of pantothenase from Pseudomonas fluorescens.

Protection of pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) against thermal inactivation by ligands was studied. The protective properties of 162 different small-molecule compounds were tested to find the most effective compounds. The best protective results were obtained with oxamate, 3-oxoglutarate, 2-oxomalonate, oxalate and oxaloacetate, in that order. The protection constants (eta) of certain metabolic intermediates were estimated using differential thermal inactivation. Generally they were observed to be lower than the corresponding inhibition constants. The deltaH of the oxalate binding derived from eta values was --165 kJ/mol. Normally, the activation energy of thermal inactivation of pantothenase in the absence of protective compounds is 220 kJ/mol, and the protective ligands enhance the measured activation energies of inactivation.

Purification and properties of pantothenase from Pseudomonas fluorescens.

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.

The computation of hyperbolic dependences in enzyme kinetics.

A computer program aimed at analysing results following Michaelis-Menten kinetics can be used unmodified in the treatment of other kinetic results provided that the kinetic equations in these cases can be written in the form of the Michaelis-Menten equation. A list is presented of the parameters to be set instead of substrate concentration and reaction rate, and of constants replacing Km and V, if such a program is applied in analysing enzyme inhibitions, activations and pH-dependences.