Reactions of the sulfhydryl groups of alanyl-tRNA Synthetase.

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PPi exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn2+, which is bound to the enzyme in the absence of ATP.

Partial reactions of aminoacyl-tRNA synthetases as functions of pH.

The effect of pH on the properties of the partial reactions of arginyl-tRNA synthetase of E. coli has been investigated. V max of pyrophosphorolysis of arginyl adenylate has a pH optimum at pH 6.1, whereas V max of the transfer of arginine to tRNA has a pH optimum of 8.2. These values correlate with the pH optima of the ATP:PPi exchange and the overall esterification reaction, respectively. Only the pyrophosphorolysis reaction requires a divalent cation; transfer proceeds in the presence of EDTA. Inorganic pyrophosphate inhibits the transfer reaction to an extent independent of the concentration of tRNA; the maximum inhibition is a function of pH, corresponding to the relative rate of pyrophosphorolysis of the common intermediate compared with the rate of transfer. These results show that different groups on the enzyme participate in the rate-limiting steps of the two partial reactions and that these partial reactions have properties consistent with their participation in the overall esterification of arginine with tRNA.

Evidence for single mechanism for aminoacyl-tRNA synthetases including aminoacyl adenylates as intermediates.

The rate of transfer of amino acid from enzyme-bound aminoacyl adenylate to tRNA has been compared with the rate of esterification of free amino acid. The approach of Lövgren et al. (Lövgren, T. N. E., Heinonen, J., and Loftfield, R. B. (1975) J. Biol. Chem. 250, 3854-3860) was used, with 14C in the aminoacyl adenylate and 3H in the free amino acid and with both the lysine and isoleucine systems of Escherichia coli. In both systems kinetic analyses show more rapid transfer from the preformed enzyme complex when interference by the back reaction with inorganic pyrophosphate was eliminated. Parallel experiments, in which the amount of enzyme complex was measured, confirmed that aminoacyl adenylate is an intermediate in both systems. No evidence was found for an alternative mechanism.

Primary structure of tRNA-Lys of E. coli B.

The primary structure of tRNALys of E. coli was determined by use of [32P]-tRNA. The sequence is pGGGUCGUUAGCUCAGDDGGDAGAGCAGUUGACUmam5-s2-UUU-t6AApsiCAAUUGm7GXCGCAGGTpsiCGAAUCCUGCACGACCCACCA. No s4-U was detected in position 8. No other lysine tRNA was detected but the existence of another species has not been ruled out.

Kinetic demonstration of the intermediate role of aminoacyl-adenylate-enzyme in the formation of valyl transfer ribonucleic acid.

The question whether aminoacyl-tRNA synthetases act in a stepwise or in a concerted mechanism has been investigated kinetically with the valine enzyme of Escherichia coli, which had been used in previous studies by others who concluded that the physiological mechanism is concerted. An exchange between aminoacyl-tRNA and tRNA, dependent upon AMP, was studied. PP-i inhibits this exchange completely in the presence of Mg2+ and AMP but in the absence of added Mg2+ or with dAMP as the nucleotide the inhibition by PP-i is only partial; this is compatible with a stepwise, not a concerted, reaction. Exchange of isotopically labeled substrates in a system at chemical equilibrium also shows effects of substrate concentrations on rates in agreement with the predictions of a stepwise mechanism.

The role of divalent cations in the reactions of valyl transfer ribonucleic acid synthetase of Escherichia coli. Effects of spermine and ethylenediaminetetraacetate.

We have analyzed the function of spermine in the aminoacylation of tRNA-Val by the valyl-tRNA synthetase of Escherichia coli. Our results indicate that Mg2+ is required for the aminoacylation reaction as well as for the ATP-PP-i exchange catalyzed by this enzyme. The apparent stimulation by spermine is a function of the tRNA used, which appears to contain bound cations even after dialysis against 10 minus 4 M EDTA. Higher concentrations of EDTA totally abolish spermine-stimulated esterification of tRNA-Val.

Deamidation in vivo of an asparagine residue of rabbit muscle aldolase.

Microheterogeneity of rabbit muscle aldolase is caused by deamidation in vivo of an asparagine residue near the C-terminus of each subunit. Isotopic labeling of a peptide containing the asparagine residue at various time intervals before isolation of aldolase permits estimation of the half-time for the deamidation as about 8 days, which is about the time estimated for the half-life of the enzyme in vivo. It is concluded that the aldolase as genetically determined is a tetramer, designated alpha(4), that undergoes random deamidation to form alpha(3)beta, alpha(2)beta(2), and alphabeta(3) species as intermediates in the formation of beta(4), the species in which all of the specific asparagine has been deamidated. Isoelectric focusing data indicate that the subunits do not exchange appreciably in vivo.