SOME PROPERTIES OF RAT LIVER GLYCYL-RNA SYNTHETASE

Abstract

Some properties of rat liver glycyl-RNA synthetase are described. The fact that methylamine competitively inhibited the glycine-dependent ATP–32PP exchange catalyzed by the enzyme suggested that glycine might be bound to the enzyme at an anionic site. The KIfor methylamine was 6.3 mM as compared with the KMfor glycine of 0.60 mM. The effects of pH on the glycine-dependent ATP–32PP exchange and on glycyl-RNA formation were very similar and indicated that these activities depended on the presence in the active center of a titratable group with a pK in the region of pH 6.2–6.7. These results suggested that an uncharged imidazole group of a histidine side chain might be necessary for activity. Further evidence for histidine in the active center came from studies on photoinactivation of the enzyme in the presence of methylene blue. It was found that glycine, but not ATP, protected the enzyme from photoinactivation and that the presence of ATP abolished this protection. A previous finding, that the partially purified enzyme fractions which had been freed of RNA were sensitive to sulfydryl agents, was confirmed. Para-hydroxymercuribenzoate (10−5M) completely inhibited the glycine-dependent ATP–32PP exchange catalyzed by the purified enzyme. Neither glycine alone nor ATP alone afforded appreciable protection of the enzyme against inactivation by PHMB, but, if the enzyme were preincubated with both of these substrates, protection was obtained. It was found that soluble RNA, but not ribosomal RNA, also protected the enzyme against the action of this inhibitor. These results suggested that a sulfydryl group of a cysteine side chain is also present in the active center of this enzyme. A tentative proposal is put forward for the mechanism of activation of glycine based on these results and on the findings of other investigators with other amino acyl-RNA synthetases.