Isoleucyl-tRNA Synthetase from Baker’s Yeast. Influence of Substrate Concentrations on Aminoacylation Pathways, Discrimination between tRNA^IIeand tRNA^Val, and between Isoleucine and Valine

Abstract

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNA Ile two K m values each differing by a factor about five were determined; the higher values were observed at concentrations higher than ΙμΜ, the lower values below ΙμΜ isoleucine or tRNA Ile , respectively. At substrate concentrations below ΙμΜ also k^t values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNA VaI such differences could not be detected. The substrate ATP did not show any change of its K m value as far as the reaction was measurable.Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNA Ile as the third one; pyrophosphate is the first product to be released, isoleucyltRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From fc^ and K m values and from AMP formation rates discrimination factors for discrimination between tRNA{l e and tRNAy al as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant.The two different K m values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.

On the non‐liner Eadie plots of the tRNA kinetics and non‐liner Dixon plots of the PPi inhibition kinetics of the aminoacyl‐tRNA synthetases

“…Another abnormality was found with the isoleucyl-tRNA synthetase from Escherichiu coli, where the Eadie plots of the tRNA kinetics were curved downwards. Similar non-linear Eadie plots have been found also for the tRNA kinetics of the Ile-tRNA synthetase from yeast [6].…”

“…are also straight lines when ATP or the amino acid is the variable substrate. If curves are obtained in measurements [6,81, they must be caused by features not taken into account in Scheme 1 .…”

“…a) Curvature in the Eadie plots of the tRNA kinetics does not prove the existence of two binding sites for tRNA [6] although, naturally, two binding sites can cause a curved Eadie plot, for example in many cases of two identical subunits [3], with cooperativity between subunits or half-of-the-sites activity detected in some aminoacyl-tRNA synthetases 1321. The direction of curvature is determined by the relation k+,/k+,, but also by the efficiency of binding of the other substrates in the absence and presence of tRNA.…”

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNAIle-C-C-A(3'NH2)

“…This enzyme mixture ' Table 1. Specific activities of ''C-labelled amino acids, solvent systems for thin-layer chromatography, and content of isoleucine Solvent systems are (1) was used in a kinetic experiment instead of the pure enzyme (concentrations in the aminoacylation assay: 100 pM isoleucyl-tRNA and 5 pM valyl-tRNA synthetase). In misacylation of tRNA1Ie-C-C-A(3'NH2) with valine the same k,,, and K , values were observed as with the purified enzyme, indicating that the measured kinetic constants are not falsified by traces of other enzymes.…”

Isoleucyl‐tRNA synthetase from baker's yeast and from Escherichia coli MRE 600

“…As pointed out in our preceding work [l] in the aminoacylation of tRNA"'-C-C-A(3'NH2) the reaction products are aminoacyl amides which are stable against enzymatic hydrolysis; thus, in this reaction, hydrolytic proof-reading can only take place before transfer of the amino acid to the tRNA. In contrast, aminoacylation of tRNA""-C-C-A results in generation of aminoacyl esters which can be hydrolysed by the enzyme [5] ; therefore in acylation of the natural substrate tRNA""-C-C-A a post-transfer proof-reading step also has to be taken into account, by which misacylation products are hydrolysed before released from the enzyme [5]. Both proofreading factors multiply to the resulting proof-reading factor n' of the complete process: 171 = n, .…”