Journal
BIOCHEMISTRY
Volume 42, Issue 51, Pages 15102-15113Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0355701
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- NIGMS NIH HHS [GM62491, GM54899, R01 GM054899] Funding Source: Medline
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Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K-3 = 450 +/- 180 muM) and threonine from the E.ATP complex (K-4' = 135 muM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thrsimilar toAMP complex, affording the dissociation constant for PPi (K-6 = 170 +/- 50 muM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K-4[HNV] = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.
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