Valyl-tRNA Synthetase from Yellow Lupin Seeds: Hydrolysis of the Enzyme-Bound Noncognate Aminoacyl Adenylate as a Possible Mechanism of Increasing Specificity of the Aminoacyl-tRNA Synthetase

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

Abstract

Lupin valyl-tRNA synthetase catalyzes the hydrolysis of ATP to AMP and pyrophosphate in the absence of tRNA and in the presence of the following amino acids: cysteine (KM = 22.5 mM; Vrel = 1), threonine (KM = 16 mM; Vrel = 0.2), alanine (KM = 27 mM; Vrel = 0.3), serine (KM = 14 mM; Vrel = 0.12), and α-aminobutyrate (KM = 1.5 mM; Vrel = 0.1). This activity is due to hydrolysis of the enzyme-bound noncognate aminoacyl adenylates. The enzyme-bound valyl adenylate is apparently protected by the enzyme and slowly decomposes with a rate constant of 0.018 min-1, which is 720 times slower than the rate constant of hydrolysis of valyl-tRNA synthetase-bound cysteinyl adenylate(k = 13 min-1). The KM value for ATP in the ATP pyrophosphohydrolase reaction is 9 αM and does not depend on the nature of the amino acid. This value is one order of magnitude lower than the KM value for ATP in the reaction of tRNA aminoacylation with valine (ATM = 100 αM). The cysteine-dependent ATP pyrophosphohydrolase activity of valyl-tRNA synthetase in the absence of tRNA exhibits pH optimum between 9.5 and 10.5 in glycine-NaOH buffer, is moderately sensitive to KC1 (50% and 60% inhibition at 150 and 300 mM KCl, respectively), is not affected by 0.1-2 mM spermine, and exhibits a temperature dependence with an Arrhenius energy of activation, Ea = 64.5 kJ., which is the same as that for tRNA aminoacylation with valine. Transfer RNA stimulates the reaction to a degree depending on the nature of amino acid, ATP concentration, pH and kind of buffer used, and KC1 and spermine concentrations. Changes of magnesium ion concentration in the range 0.25-10 mM do not affect the stimulation. The degree of stimulation by tRNA of the ATP pyrophosphohydrolase activity also does not depend on temperature. The tRNA apparently acts as an allosteric activator, which binds to the enzyme with Kdiss = 20 nM and increases KM for ATP from 9 to 100 αM. The ATM for amino acids is either not affected (KM = 28 mM for alanine and 14 mM for serine) or slightly affected (KM = 9.6 mM for cysteine and 8.6 mM for threonine) by the presence of tRNA. Transfer RNA devoid of amino acid acceptance by periodate oxidation, albeit able to bind to the enzyme as well as intact tRNA, cannot produce these effects and does not inhibit the ATP pyrophosphohydrolase activity of valyl-tRNA synthetase. Lupin valyl-tRNA synthetase apparently rejects noncognate amino acids at the level of aminoacyl adenylate. The contribution of this pathway of rejection to the overall rejection of noncognate amino acids in the presence of tRNA is calculated to be 25%, 19%, 12%, 2.5% and 2% for cysteine, alanine, serine, threonine, and and α-aminobutyrate, respectively.

Original languageEnglish (US)
Pages (from-to)5071-5078
Number of pages8
JournalBiochemistry
Volume19
Issue number22
DOIs
StatePublished - 1980
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Fingerprint

Dive into the research topics of 'Valyl-tRNA Synthetase from Yellow Lupin Seeds: Hydrolysis of the Enzyme-Bound Noncognate Aminoacyl Adenylate as a Possible Mechanism of Increasing Specificity of the Aminoacyl-tRNA Synthetase'. Together they form a unique fingerprint.

Cite this