Aminoacylation of Escherichia coli Valine Transfer Ribonucleic Acid by Neurospora crassa Phenylalanyl Transfer Ribonucleic Acid Synthetase in Tris(hydroxymethyl)aminomethane Hydrochloric Acid and Potassium Cacodylate Buffers: EFFECT OF SALTS, DIMETHYL SULFOXIDE, ETHANOL, AND 2-MERCAPTOETHANOL

Abstract

Abstract Aminoacylation of valine transfer RNA from Escherichia coli by phenylalanyl-tRNA synthetase from Neurospora crassa is extremely sensitive to assay conditions. In Tris buffer under optimal conditions, the final yield of phenylalanyl-tRNAval is a function of enzyme concentration, and aminoacylation is incomplete even at very high enzyme concentrations. At low enzyme concentrations, the final yield of phenylalanyl-tRNAval is increased 10-fold by 20% dimethyl sulfoxide. Ethanol (10%) also increases the extent of aminoacylation, whereas NaCl, NH4Cl, and 2-mercaptoethanol all lead to decreased final yields. When cacodylate buffer is used in place of Tris buffer, the pH optimum for aminoacylation is shifted from above 8.1 to 6.3, and various kinetic properties of the aminoacylation reaction are changed. At low enzyme concentrations over 10 times more phenylalanyl-tRNAval is formed in cacodylate buffer than is formed in Tris buffer. Dimethyl sulfoxide (20%) and all concentrations of 2-mercaptoethanol, ethanol, NaCl, and NH4Cl inhibit aminoacylation in cacodylate buffer. These results emphasize that buffers are not necessarily inert compounds merely maintaining the pH of a reaction mixture and that the first step in the study of a heterologous aminoacylation reaction should be the careful optimization of reaction conditions.