Kinetics of acyl-tRNA complexes of Escherichia coli phenylalanyl-tRNA synthetase. A conformational change is rate limiting in catalysis

Abstract

Kinetics of complex formation between phenylalanyl-tRNA synthetase and Phe-tRNAPhe have been measured by the stopped-flow technique. Either the protein intrinsic fluorescence or the fluorescence of the added indicator 6-(p-toluidinyl)naphthalene-2-sulfonate was observed. Identical results were obtained with each method. Acyl-tRNAs with variable structures of the acyl and tRNA moieties were examined. Kinetics were measured as a function of pH and at different ionic strengths. Kinetic constants were compared with those of enzymatic phenylalanylation of tRNAPhe. The results are as follows. (1) Phe-tRNAPhe binds to phenylalanyl-tRNA synthetase in two, mutually exclusive types of complexes, one at the tRNA-specific binding site of the enzyme and the other in a region which involves the Phe-specific binding site of the enzyme [Holler, E. (1980) Biochemistry 19, 1397-1402]. The Phe site directed association includes a conformational change of the complex that is rate limiting. (2) The conformational change and catalytic tRNA aminoacylation follow similar values of rate constants irrespectively of pH and ionic strength. It is concluded that aminoacylation is limited by the kinetics of a conformational change of the nascent enzyme-Phe-tRNAPhe complex. (3) The nature of Phe site directed binding was probed by variation of the structure of Phe-tRNAPhe. Both the acyl and the tRNA moieties are recognized by the enzyme. Of the acyl moiety, only the phenyl ring but not the amino group is essential for binding. The amino group can be acetylated or replaced by a hydroxyl group. Protonation of the amino group results in loss of Phe site directed binding. It gives a pKa = 6.9, which is close to that for protonation of a phenylalanine ester. (4) Rate constants were only slightly affected by addition of 200 mM NaCl at pH 7.5, indicating that the contribution by electrostatic forces was probably minimal. Mg2+ ions were essential for Phe site directed binding. Complexation of enzyme, Phe-tRNAPhe, and Mg2+ either was random or was at preequilibrium with the conformational change. (5) Binding of Phe-tRNAPhe at the tRNA-specific site of the enzyme was studied in the presence of in situ synthesized phenylalanyl adenylate. The reaction was bimolecular with rate constants of 50 microM-1 s-1 and 15 s-1 for association and dissociation, respectively.

Resampling and Editing of Mischarged tRNA Prior to Translation Elongation

“…To prevent hydrolysis of Tyr-tRNA Phe , an editing-defective PheRS variant (βA356W) was employed. Phe-tRNA Phe and Tyr-tRNA Phe showed no significant differences in any of the kinetic parameters measured (Figure 2C), and the constants were comparable to those previously measured for E. coli PheRS binding of Phe-tRNA Phe (Baltzinger and Holler, 1982). …”

“…In class II aaRSs such as AlaRS, PheRS, and ProRS, ami-noacyl-tRNA release is rapid and not rate limiting during aminoacylation, while the rate of aminoacylation by class I enzymes is limited by product release (Baltzinger and Holler, 1982; Ibba et al, 1995; Zhang et al, 2006). To examine whether these class-specific differences in activity also extend to the dissociation and rebinding of mischarged tRNAs, we investigated the impact of EF-Tu on editing by LeuRS (class I) (Lincecum et al, 2003) and ProRS (class II) (Beuning and Musier-Forsyth, 2000).…”

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

“…The concentration dependence of k A yields a second-order rate constant of 1 Â 10 8 M À1 s À1 , which is less than the diffusion controlled limit for macromolecular interactions. 42 Similar¯uorescence transitions upon formation of RNA-protein complexes occurring at equally fast rates have also been observed in substrate binding by aminoacyl-tRNA synthetases (4 Â 10 7 M À1 s À1 ), 23,24 binding of the restriction enzyme EcoRV to its substrate (5 Â 10 7 M À1 s À1 ), 70 binding of the termination factor Rho to RNA Figure 10. Kinetic scheme for single turnover cleavage of R1.1 RNA by RNase III.…”

“…In addition to advancing our understanding of the RNase III mechanism the present study also provides, for the ®rst time, an assessment of conformational changes during substrate binding. Conformational changes appear to be a common aspect of RNA-protein interactions, and stopped-ow¯uorescence has been successfully used to study RNA-protein dynamics in several cases, 23,24,65,66 as well as for understanding RNA dynamics. 67 ± 69 We ®nd that there are conformational transitions associated with both binding and catalysis by RNase III (Figure 10).…”

Effects of the ligands of beef tryptophanyl-tRNA synthetase on the elementary steps of the tRNATrp aminoacylation

“…Such a conformational change has been observed during the aminoacylation reaction for several synthetases (Riesner et al, 1978;Kern & Gangloff, 1981;Holler et al, 1981; Baltzinger & Holler, 1982b;Beresten et al, 1983;Ferguson & Yang, 1986a) and for tRNA (Renaud et al, 1981;Lefevre et al, 1981;Yamashiro-Matsumara & Kawata, 1981;Ferguson & Yang, 1986b;Pelka & Schulman, 1986). It may be limiting (Baltzinger & Holler, 1982a). Finally, it is also conceivable that tRNATrp perturbs the activation step strongly enough to make this step a partially limiting factor in the overall reaction.…”