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
Abstract:Error rates in discrimination between cognate tRNAIle and noncognate tRNAVal in the aminoacylation reaction with isoleucine catalyzed by isoleucyl-tRNA synthetase from yeast have been investigated in three sets of experiments under different assay conditions. The overall discrimination factor was first determined by isoleucylation of tRNAVal/tRNAIle mixtures. In the second set of experiments, the number of AMP molecules formed per Ile-tRNA in the cognate and noncognate reactions was measured. The higher AMP fo… Show more
“…Although these rate constants were determined under different conditions and for an enzyme from another source, they may also indicate the order of magnitude of rate constants belonging to the yeast enzyme. Under standard conditions (pH 7.65,O.l M KCI) we have found a sequential ordered ter-ter mechanism for aminoacylation of tRNA"'-C-C-A with isoleucine by isoleucyl-tRNA synthetase from yeast [24] (Scheme 2). The kca, value was 0.83 s-'.…”
Specificity with regard to amino acids in aminoacylation of tRNAIle‐C‐C‐3′dA by isoleucyl‐tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2= 180–1700), indicating that at same amino acid concentrations isoleucine is 180–1700 times more attached to tRNAIle‐C‐C‐3′dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2= 10 000–30 000). D2 values of the other amino acids are in the range of 2000–10 000.
Recognition of most amino acids is achieved in a four‐step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof‐reading steps occur on the pre‐ and the post‐transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post‐transfer proof‐reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post‐transfer proof‐reading step are lacking.
The role of the terminal hydroxyl groups of the tRNA for post‐transfer proof‐reading is assigned to a simple neighbouring group effect. No preference for the 2′ or 3′ position in proof‐reading can be postulated.
“…Although these rate constants were determined under different conditions and for an enzyme from another source, they may also indicate the order of magnitude of rate constants belonging to the yeast enzyme. Under standard conditions (pH 7.65,O.l M KCI) we have found a sequential ordered ter-ter mechanism for aminoacylation of tRNA"'-C-C-A with isoleucine by isoleucyl-tRNA synthetase from yeast [24] (Scheme 2). The kca, value was 0.83 s-'.…”
Specificity with regard to amino acids in aminoacylation of tRNAIle‐C‐C‐3′dA by isoleucyl‐tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2= 180–1700), indicating that at same amino acid concentrations isoleucine is 180–1700 times more attached to tRNAIle‐C‐C‐3′dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2= 10 000–30 000). D2 values of the other amino acids are in the range of 2000–10 000.
Recognition of most amino acids is achieved in a four‐step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof‐reading steps occur on the pre‐ and the post‐transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post‐transfer proof‐reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post‐transfer proof‐reading step are lacking.
The role of the terminal hydroxyl groups of the tRNA for post‐transfer proof‐reading is assigned to a simple neighbouring group effect. No preference for the 2′ or 3′ position in proof‐reading can be postulated.
“…The possible
evolutionary order of these kinetic mechanisms is given in Fig. 1 (Murray et al 2002; Vitte 2015; Benner 1989; Wang and Wu 2007; Zuccotti et al 2001; McClard et al 2006; Yu et al 2014; Freist and Sternbach 1984; Celeste et al 2012; Kim and Kang 1994; Menefee and Zeczycki 2014; Vergnolle et al 2013). Fig.…”
Section: Need For Different Kinetic Mechanismsmentioning
confidence: 99%
“…tRNAVal is aminoacylated in rapid equilibrium random ter–ter order via a bi–bi uni–uni ping-pong mechanism with a rapid equilibrium segment and via two bi–uni uni–bi ping-pong mechanisms. It is assumed that assay conditions can be regarded as a stepwise approximation of physiological conditions and that considerable changes in error rates, up to one order of magnitude, may be possible in vivo (Freist and Sternbach 1984). Numerous steady-state kinetic studies have examined the complex catalytic reaction mechanism of multifunctional enzymes, such as pyruvate carboxylase.…”
Section: Need For Different Kinetic Mechanismsmentioning
This review paper discusses the reciprocal kinetic behaviours of enzymes and the evolution of structure–function dichotomy. Kinetic mechanisms have evolved in response to alterations in ecological and metabolic conditions. The kinetic mechanisms of single-substrate mono-substrate enzyme reactions are easier to understand and much simpler than those of bi–bi substrate enzyme reactions. The increasing complexities of kinetic mechanisms, as well as the increasing number of enzyme subunits, can be used to shed light on the evolution of kinetic mechanisms. Enzymes with heterogeneous kinetic mechanisms attempt to achieve specific products to subsist. In many organisms, kinetic mechanisms have evolved to aid survival in response to changing environmental factors. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzyme promiscuity is defined as adaptation to changing environmental conditions, such as the introduction of a toxin or a new carbon source. Enzymes with broad substrate specificity and promiscuous properties are believed to be more evolved than single-substrate enzymes. This group of enzymes can adapt to changing environmental substrate conditions and adjust catalysing mechanisms according to the substrate’s properties, and their kinetic mechanisms have evolved in response to substrate variability.
“…Though recent study on class I CysRS shows increased rate of aminoacylation in the presence of the elongation factor EF-TU, 11 earlier studies of class I IleRS do not exhibit any change in the rate in the presence of EF-TU. 21,22 The reason for this behavior is not clear.…”
Aminoacyl-tRNA synthetases (aaRS) catalyze the bimolecular association reaction between amino acid and tRNA by specifically and unerringly choosing the cognate amino acid and tRNA. There are two classes of such synthetases that perform tRNA-aminoacylation reaction. Interestingly, these two classes of aminoacyl-tRNA synthetases differ not only in their structures but they also exhibit remarkably distinct kinetics under pre-steady-state condition. The class I synthetases show initial burst of product formation followed by a slower steady-state rate. This has been argued to represent the influence of slow product release. In contrast, there is no burst in the case of class II enzymes. The tight binding of product with enzyme for class I enzymes is correlated with the enhancement of rate in presence of elongation factor EF-TU. In spite of extensive experimental studies, there is no detailed theoretical analysis that can provide a quantitative understanding of this important problem. In this article, we present a theoretical investigation of enzyme kinetics for both classes of aminoacyl-tRNA synthetases. We present an augmented kinetic scheme and then employ the methods of time-dependent probability statistics to obtain expressions for the first passage time distribution that gives both the time-dependent and the steady-state rates. The present study quantitatively explains all the above experimental observations. We propose an alternative path way in the case of class II enzymes showing the tRNA-dependent amino acid activation and the discrepancy between the single-turnover and steady-state rate.
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