Seryl Transfer Ribonucleic Acid Synthetase from Escherichia coli

The binding of l-serine and ATP to seryl-tRNA synthetase from Escherichia coli was examined by equilibrium dialysis. The gel filtration method of Hummel and Dryer ((1962) Biochim. Biophys. Acta 63, 530) was used to estimate the binding of the enzyme to tRNAser. The molar ratio of substrate bound to the enzyme in each instance was approximately 2:1, suggesting that each subunit has one binding site for each of the substrates. Several sulfhydryl reagents were employed to evaluate the role of the thiol groups in the synthetase. When the enzyme was treated with high concentrations of 5,5'-dithiobis(2-nitrobenzoate) or [14C]iodoacetamide, only 1 of the 10 cysteinyl residues per subunit was modified and the enzyme retained full aminoacylation activity. On the other hand, reaction with equivalent concentrations of [14C]iodoacetate appeared to be a two-step process. The initial rate of carboxymethylation was characterized by a hyberbolic curve which slowly progressed to a point where 0.75 to 0.9 carboxymethyl groups were incorporated per subunit, with no detectable inactivation. At this point, the reaction rate increased in an exponential fashion and was accompanied by a nonlinear loss of enzyme activity. Similarly, titration of seryl-tRNA synthetase with p-mercuribenzoate (PMB) was characterized by a linear inactivation-inhibitor concentration relationship which required 20 moles of PMB per mole of enzyme for complete inactivation, suggesting that sulfhydryl group modification with PMB is an all-or-none event. It is proposed that the thiol groups of the seryl-tRNA synthetase possess a structural rather than a catalytic role. Seryl-tRNA synthetase was covalently linked to cyanogen bromide-activated Sepharose and this material was used for chromatography of tRNA from E. coli. A one-step purification of serine transfer RNA was achieved. The stable Sepharose-synthetase conjugate has also been used to study the effects of reagents such as urea on the dissociation of the enzyme.