Isoleucyl‐tRNA Synthetase

Abstract

Fluorescence properties of purified isoleucyl-tRNA synthetase isolated from Escherichia coli B have been studied. No changes in the quantum yield, energy or polarisation of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L-isoleucine.In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme-ligand complexes. Values have been obtained for the complexes between enzyme and L-isoleucine (&iSs = 25 pM), L-valine (KdiS8 = 300 pM), ATP (Kdiss = 150 pM) and PPi (&iss = 200 pM) at 25". The effects of ionic strength, and the temperature dependence and urea concentration dependence of L-isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and L-isoleucine together. The magnesium ion concentration dependence of this effect (half-point about 200 pM) and the equilibrium constant for L-isoleucine activation (2 pM) have both been measured.The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound L-isoleucyladenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.The reactions catalysed by isoleucyl-tRNA synthetase are usually represented by the following equilibria :Enzyme + ATP + Ile ,-' Enzyme-AMP-Ile Yarus and Berg, studying interactions between isoleucyl-tRNA synthetase and tRNAIle, have found that the rates a t which complexes between enzyme and tRNA form and dissociate are both enhanced in the presence of L-isoleucine [l,S]. They explain their results on the hypothesis that isoleucyl-tRNA synthetase adopts several conformations during a single catalytic cycle [l]. A problem in comparing and interpreting the results of these and other groups is that the physical conditions used are often widely different.

On the metal‐ion coordinating properties of the 5′‐monophosphates of 1, N6‐ethenoadenosine (ɛ‐AMP), adenosine and uridine

“…The results are listed in Table 1 together with some related data [17 -201. The value of 4.23 for pKE2(E-AMP) agrees well with the value of 4.3 which was determined earlier [21]. The predominant site of protonation in the &-adenine moiety is N-6 [13,16,22] while in the parent adenine moiety it is N-1 [18].…”

Dihydropteridine Reductase

“…However, when incubated in the presence of its ligand, NADH, the enzyme was to a certain extent protected against inactivation. Ligand protection of an enzyme against inactivation has previously been used for determination of the dissociation constant for enzyme -ligand complex [3].…”

“…The ligand protection method is based on the fact, that a n enzyme to some extent is protected against denaturation, when a ligand is bound to it. Thus, a relationship exists between the dissociaton constant for the ligand -enzyme complex and the denaturation rate of the enzyme [3].…”

Aminoacyl‐tRNA Synthetases: Some Recent Results and Achievements

“…O n the one hand, there are methods that do not involve the isolation of complexes; for instance, studies of the effect of substrates on the thermostability of the synthetases (79)(80)(81), on resistance to proteolysis (81), or the action of other denaturing factors (82) or studies of the effect of substrates on the fluorescence of the enzyme (28,38,(83)(84)(85)(86)(87). Fluorescence techniques are used to investigate such characteristics of the enzyme as quantum yield, position and halfwidth of the fluorescence maximum of the aromatic amino acid residues of the enzyme protein (mainly tryptophan) (28,38,83), or the kinetics of the fluorescence quenching in the presence of denaturing agents (85). Both approaches-inactivation studies and fluorescence techniques-have been applied to study the binding of all three substrates.…”

“…However, studies of the complexes alone cannot prove any reaction route because direct information to this end can be obtained only by kinetic methods. Moreover, it has been demonstrated for a number of synthetases that each substrate alone (in the absence of other substrates) is bound by the enzyme (28,38,85,88). At the same time the evidence obtained by investigation of the complexes does not contradict the results of kinetic studies: it was found by means of gelfiltration techniques that ATP forms complexes with leucyl-tRNA (125), threonyl-tRNA (124), and tryptophanyl-tRNA (1 17,118) synthetases.…”