RATE-DETERMINING STEP IN AMINOACYLATION OF TRNA Shapiro, A. L., and Maizel, J. V. Jr. (1969), Anal. Biochem. Sun, T. -T., Bollen, A., Kahan, L., and Traut, R. R. (1974), Thomas, J. O., and Kornberg, R. D. (1975), Proc. Natl. Acad. Vogel, O., and Henning, U. (1971), Eur. J. Biochem. 18, Vogel, O., Hoehn, B., and Henning, U. (1972), Proc. Natl. Wang, K., and Richards, F. M. (1974), J. Biol. Chem. 249, 29, 505-514. 103-1 15. Biochemistry 13. 2334-2340. Acad. Sci. U.S.A. 69, 1615-1619. Sci. U.S.A.72. 2626-2630. 8005-8018. The Mechanism of the Aminoacylation of Transfer Ribonucleic Acid: EnzymeeProduct Dissociation Is Not Rate Limiting? Timo Nils E. Lovgren,t,l Andrzej Pastuszyn, and Robert B. Loftfield* ABSTRACT: It has been proposed that the rate-limiting step in the synthesis of aminoacyl-tRNA is the rate at which the product dissociates from the enzyme. The experimental evi- dence supporting this hypothesis comes from work at low pH and low temperature (although the reaction has been argued to have the same mechanism under physiological conditions). We have reexamined the binding assay by which M. Yarus and P. Berg (1969) (J. Mol. Biol. 42, 171-189) measured the kd for dissociation of Enz-(Ile-tRNA). We find that when overall reaction and dissociation are measured under identical con- ditions the two rates are not the same. Moreover, while an in- crease in ionic strength greatly stimulates dissociation, the same increased ionic strength slows aminoacylation. Spermine accelerates overall aminoacylation without affecting disso- ciation. Because any change in a rate-limiting step must, by definition, cause a parallel change in the overall reaction, these observations prove that under these conditions the synthesis of Ile-tRNA is not limited by the rate of dissociation of Enz. (Ile-tRNA). Entirely similar observations were made for the dissociation of Enz-(Val-tRNA) and the overall synthesis of Val-tRNA at 0 OC, pH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacy- lation was shown not to be saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged I t has been proposed that the rate-limiting step in the syn- thesis of several aminoacyl-tRNAs (AA-tRNA1) is the sep- From the Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, New Mexico 871 3 1. Received De- cember 31, 1975. This work has been supported by United States Public Health Grant CA-08000 and by American Cancer Society Grant NP103D. A preliminary report of this work has been made (Pastuszyn et al., 1974). * Present address: Institutionen for Biokemi och Farmaci, Abo Akademi, 20500 Abo, Finland. $Recipient of a travel stipend from Finlands Akademi Naturveten- skapliga Kommissionen. I Abbreviations used are: tRNAAA,unesterified transfer ribonucleic acid specific for a particular amino acid; AA-tRNh, transfer ribonucleic acid esterified with its specific amino acid; Enz, free enzyme (amino acid:tRNA ligase specific for a particular amino acid (EC 6.1.1)); AA-AMP aminoacyl adenylate; Enz.(tRNA1Ie), Enz.(Val-tRNA), Enz.(AA-AMP), etc., ligase bound to tRNA1Ie,to Val-tRNA, to ami- noacyl adenylate etc., respectively; EDTA, (ethylenedinitri1o)tetraacetic acid; DEAE, diethylaminoethyl; GSH, reduced glutathione. substrate tRNA and with product Val-tRNA. E. W. Eldred and P. R. Schimmel (( 1972) Biochemistry 11, 17-23) report that the formation of Ile-tRNA proceeds at two rates: (a) k = 2 X s-l until the enzyme is saturated with the first mole of product, and (b) k = 2 X s-l for subsequent cycles. We did not observe this behavior at any pH or temperature with four different amino acid:tRNA ligases. Because aminoacy- lation proceeds more rapidly than “dissociation” under some conditions, we believe that the binding assay measures not only enzyme-product dissociation but also other slower reactions such as aggregation or disaggregation of Enz.(AA-tRNA). In conjunction with recent studies from other laboratories, this work makes it unlikely that enzyme-product dissociation is the rate-limiting step in the synthesis of aminoacyl-tRNA either at low temperature and pH or under more nearly physiological conditions. From the effect of salt, it would appear that the rate of aminoacylation of tRNA is largely limited by the rate or extent of formation of Enz.(tRNA) (Loftfield, R. B., and Eigner, E. A. (1967), J. Biol. Chem. 242, 5355-5359). Using the binding assay of M. Yarus ((1972) Biochemistry 11, 2050-2060), we find the K,,, for Enz.(Ile-tRNA) varies lin- early with the Debye-Huckel function at ionic strengths of 0.1-0.4 from los to lo6. aration of the product from the enzyme (Yarus and Berg, 1969; Htlbne et al., 1971; Eldred and Schimmel, 1972, 1973). If a particular step is rate limiting, a change in rate of that step should change the overall reaction in a parallel way. It seemed probable that raising the ionic strength would accelerate dissociation (Pingoud et al., 1973; Krauss et al., 1973; Loftfield and Eigner, 1967); therefore, we sought to determine, under Yarus and Berg’s binding assay conditions, the effect of ionic strength on the overall reaction. In general under other conditions, it is known that higher ionic strengths slow aminoacylation reactions (Loftfield and Eigner, 1967; Taglang, et al., 1970; Smith, 1969; Holten and Jacobson, 1969; Yarus, 1972; Loftfield, 1972). The only reported exception to this generalization involves the tRNA and enzymes derived from halophilic bacteria (Griffiths and Bayley, 1969). Simi- larly, it was likely that spermine would accelerate the overall reaction (Igarashi et al., 1971; Pastuszyn and Loftfield, 1972), so we examined the effect of spermine on dissociation. BIOCHEMISTRY, VOL. 15, NO. 12, 1976 2533