Mitochondrial phenylalanyl t-RNA synthetase from yeast: formation of enzyme-substrate complexes shown by heat or SH reagent inactivation

Abstract

The binding of substrates to purified mitochondrial phenylalanyl-tRNA synthetase from yeast was examined using the kinetics of heat or p-hydroxymercurybenzoate inactivation. Individually magnesium chloride and each of the substrates protect the enzyme against thermal denaturation and p-hydroxymercurybenzoate inhibition. No enzyme protection is observed with ATP alone against p-hydroxymercurybenzoate inhibition. The combinations of the various substrates induce a synergistic protection effect. Protection constants of 31 microM and 0.3 microM were found for L-Phe and mt tRNAPhe respectively, from heat inactivation studies. The inhibition of the enzyme activity by p-hydroxymercurybenzoate can be reverted by 2-mercaptoethanol or dithiothreitol.