Arginyl-transfer ribonucleic acid synthetase of Bacillus stearothermophilus. Purification and kinetic analysis.
Arginyl-tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified 880-fold to apparent homogeneity as demonstrated by electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight is 59 000 as confirmed by Sephadex G-100 and by sucrose gradient ultracentrifugation. The enzyme is monomeric, no subunits were detected. Its cognate tRNA induces an apparent increase in molecular weight suggesting the dimerisation of the enzyme. Nevertheless, it is not obvious that the enzyme dimer forms prior to the aminoacylation reaction catalysed by the enzyme. An ATPase activity was found associated to the synthetase but can be neglected because the ATP consumption is too low for hampering the arginyl-tRNA synthetase activity. The order of addition of substrates and release of products has been studied by measurements of initial velocity, product inhibition and dead-end inhibition. The nature of the kinetic patterns indicates that the aminoacylation reaction conforms to the classical concept of the mechanism which includes the formation of an enzyme-bound aminoacyl-adenylate as an intermediate in the first step followed by transfer of the amino acid to tRNA. The first partial reaction, measured by the ATP-32PPi exchange or AMP synthesis in the presence of ATP and arginine, requires tRNA, which is consistent with the model in which tRNAArg is an activator of the arginyladenylate synthesis.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D001126:Arginine-tRNA Ligase / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate; D046249:Transfer RNA Aminoacylation
D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases; D001126:Arginine-tRNA Ligase