Isoleucyl‐tRNA Synthetase from Baker's Yeast

Abstract

The order of substrate addition to isoleucyl-tRNA synthetase from baker's yeast has been investigated by steady-state kinetics with inhibition by four different inhibiting ATP analogs acting competitively, uncompetitively and noncompetitively with respect to ATP, namely purineriboside (= nebularin), 3'-deoxy-adenosine (= cordycepin), 8-amino-adenosine and 8-azido-adenosine 5'-triphosphates. The inhibition studies were done in the aminoacylation and in the pyrophosphate exchange reaction, the aminoacylation was investigated in the absence and presence of inorganic pyrophosphatase. Additionally, bisubstrate kinetics and product inhibition studies were carried out. The inhibition patterns indicate a multisite system with a minimum number of two sites for each of the substrates. The results of the pyrophosphate exchange studies are consistent with formation of E . Ile-AMP . ATP . Ile complexes by random addition of one ATP and one isoleucine molecule, followed by adenylate formation, subsequent release of pyrophosphate and random addition of a second molecule of ATP and isoleucine. For the aminoacylation in the absence of pyrophosphatase an ordered ter-ter mechanism is postulated; in the presence of pyrophosphatase the mechanism is random bi-uni uni-bi ping-pong. Both the pyrophosphate and the analogs of this compound such as imidodiphosphate or methylenediphosphonate can induce the enzyme to act in the ter-ter mechanism.

Isoleucyl-tRNA synthetase from bakers yeast: variable discrimination between tRNAIle and tRNAVal and different pathways of cognate and noncognate aminoacylation under standard conditions, in the presence of pyrophosphatase, elongation factor Tu-GTP complex, and spermine

“…To study a possible influence of elongation factor Tu-GTP complex on the aminoacylation mechanism, we carried out inhibition experiments with 3'-deoxyadenosine S'-triphosphate as an "indicator inhibitor" with respect to the three substrates. The inhibition patterns caused by 3'dATP were typical for the sequential ordered ter-ter mechanism (Table III) as observed in previous studies (Freist et al, 1981b); however, the Km of tRNA11' is lowered to 0.0005 mM, a value that is very similar to that one observed in experiments with modified tRNAlles (Freist & Cramer, 1983). The kal value is also considerably lowered to 0.28 s-1.…”

“…( 1) Aminoacylation of tRNAlle. In our previous work on isoleucyl-tRNA synthetase from bakers' yeast, two pathways of aminoacylation were detected depending on the presence of pyrophosphatase in the aminoacylation assay (Freist et al, 1981b); in the first case the order of substrate addition and product release is sequential ordered ter-ter, and in the second case a bi-uni uni-bi ping-pong mechanism is observed (Table III). These experiments were only carried out under the normally used assay conditions, which are optimized to obtain greatest enzyme activities.…”

“…The experiments run in the noncognate system may be of special interest because a misacylation reaction was not investigated 0006-2960/84/0423-5742$01.50/0 © 1984 American Chemical Society before by initial rate kinetics. For two different pathways of the cognate aminoacylation reaction detected by kinetic studies with isoleucyl-tRNA synthetase from yeast (Freist et al, 1981b), different fccat values had been observed, which may implicate also changes in the accuracy achieved by the enzyme.…”

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

“…Isoleucinol (DL-2-amino-3-methylpentanol) was a product of EGA Chemie (D-7924 Steinheim), 3'-deoxyadenosine 5'-triphosphate (3'-dATP) was synthesized as in ref J 14^. [8][9][10][11][12][13][14] C]Adenosine 5'-triphosphate with a specific activity of 546 mCi/mmol was purchased from Amersham Buchler GmbH (D-3300 Braunschweig), and was diluted with the ten-fold amount of unlabelled material. 333 14 C-labelled isoleucine and 14 C-labelled valine with specific activities of 342 and 275 mCi/mmol, respectl· vely were purchased from the Radiochemical Centre (Amersham, Great Britain).…”

Photoaffinity Leabeling of Chloroplastic and Cytoplasimic Leycly‐rRNA Synthetases of Euglena gracilis, by the γ‐(p‐Azidoanilidae) of ATP

“…This explanation is given for the inhibition of isoleucyl-tRNA and arginyl-tRNA synthetases of yeast by several ATP analogs which act competitively against the respective amino acid [23,24]. (b) The inhibitors really interact with the leucine subsite due to some common hydrophobic properties of the anilido-ring and leucine.…”