A role for protein--protein interactions in the maintenance of active forms of aminoacyl-tRNA synthetases.
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Amino Acyl-tRNA Synthetases↗
Biomedical subjects
H Jakubowski
Publications and source records attributed to H Jakubowski.
Yellow lupin (Lupinus luteus) aminoacyl-tRNA synthetases. Isolation and some properties of enzyme-bound valyl adenylate and seryl adenylate.
As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.
Adenosine Monophosphate↗
The mechanism of the aminoacylation of transfer ribonucleic acid. The kinetics and stoichiometry of the lysis of aminoacyl-tRNA.
It is often stated that the aminoacylation of transfer RNA proceeds in discrete steps: (formula: see article). If this is a complete description of the reaction, the reverse overall formation of ATP should not be more rapid than the formation of Enz . (AA approximately AMP). We show for four different amino acid:tRNA ligases that lysis of AA-tRNA (with PPi and AMP) to ATP is faster than lysis of AA-tRNA (with AMP only) to Enz . (AA approximately AMP). This requires that the transition state proceeds from a quaternary complex of PPi, AMP, AA-tRNA and Enz. From the law of microscopic reversibility, this requires that in the forward reaction the AA-tRNA bond be formed before PPi leaves the enzyme complex. Therefore, the forward reaction passes through the quaternary complex Enz . ATP . AA . tRNA. (In view of recent evidence of the specific requirement of two cations, the complex is accurately described as senary).
Adenosine Monophosphate↗
Incomplete aminoacylation of tRNALeu catalyzed in vitro by leucyl-tRNA synthetase from Escherichia coli B.
The extent of esterification of [14C] leucine into Escherichia coli B tRNALeu apparently depends on the concentration of leucyl-tRNA synthetase. The effect is more pronounced at pH 9.0 than at pH 7.4. When reciprocals of leucyl-tRNA concentration at plateau [aa-tRNA]-1 are plotted against reciprocals of initial velocities vo-1 of aminoacylations a straight line is obtained with a slope equal to the rate constant of non-enzymatic deacylation of leucyl-tRNA. Factors which change the stability of leucyl-tRNA, e.g. pH and temperature, also change the shape of the function [aa-tRNA]-1 vs. vo-1. The data are consistent with the idea that the rate constant of spontaneous deacylation of aminoacyl-tRNA is the factor which accounts for the dependence of the level of aminoacylation on initial velocity of aminoacylation.
Amino Acyl-tRNA Synthetases↗
Use of omega-aminohexyl-sepharose in the fractionation of Escherichia coli B aminoacyl-tRNA synthetases.
The usefulness of aminohexyl-Sepharose in purification of E. coli B aminoacyl-tRNA synthetases is presented. The purification factors for 14 synthetases lie in the range 3- to 94-fold and the recoveries of the enzymatic activity were 30-80%, depending on the enzyme.
Amino Acyl-tRNA Synthetases↗
The plant aminoacyl-tRNA synthetases. Effect of sodium chloride on tRNA aminoacylation and aminoacyl-tRNA decomposition catalysed by aminoacyl-tRNA synthetases from yellow lupin seeds.
The initial velocity and the extent of aminoacylation are affected by sodium chloride in the lupin aminoacylation systems involving serine, isoleucine, lysine, leucine, phenylalanine and valine. Pyrophosphorolysis and enzymatic hydrolysis of [14C]Val-tRNA catalysed by lupin valyl-tRNA synthetase are inhibited by sodium chloride nearly to the same extent. Evidence is presented that when a limiting amount of synthetase is used, the equilibrium of the aminoacylation reaction in the lupin valine system is determined only by the rate of aminoacylation and non-enzymatic deacylation of aminoacyl-tRNA, the former but not the latter reaction being dependent on concentration of the enzyme and monovalent salt.
Amino Acyl-tRNA Synthetases↗
Transfer RNA methyltransferases from yellow lupin seeds: purification and properties.
tRNA methyltransferases from extract of yellow lupin seeds were purified over 300-fold by the methods based on hydrophobic and affinity chromatography. However, in the most active fractions the methylating enzymes were over 2000 purified. The purified enzyme fractions catalysed the formation of 1-methyladenine and 5-methylcytosine using E. coli B and B. subtilis tRNAs as substrates and S-adenosylmethionine as the methyl donor. They were unable to methylate their own endogenous tRNA but they were capable of methylating tRNA of some other lupinus species. Whereas the patterns of methylated constituents of tRNA of some other lupinus and B. subtilis were quite similar, they differed considerably from those obtained with lupin species tRNAs. Some properties of purified methyltransferases from yellow lupin seeds have been described.
Adenine↗