Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme.cntdot.aminoacyl-tRNA complexes as a consequence of substrate specificity

Abstract

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.

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

“…Kinetics of Enzyme•Acyl-tRNA Complexes. Phenylalanyl-tRNA synthetase complexes of acyl-tRNAs were observed by their ability to decrease the fluorescence intensity of TNS (Güntner & Holler, 1979;Holler, 1980). When TNS occupies the Phe-specific binding site of the enzyme, it becomes highly fluorescent.…”

“…Modifications of tRNA. tRNAj^o, [and Phe(OH)-iRNAhnoJ was synthesized by incubation with nitrous acid as has been described (Güntner & Holler, 1979). tRNAPbe was prepared by UV irradiation as in Favre et al (1979) and was the same material as described (Holler et al, 1981a).…”

“…Phe-tRNApbc was prepared as described by Bartmann et al (1974b). Phe-tRNApbc and Ile-tRNAPbe were identical with the material used by Güntner & Holler (1979). Phenylalanylation of tRNAVal (E. coli) was performed as described (Roe et al, 1973).…”

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

“…Nonequilibrium Dialysis. The method of Colowick & Womack (1969) has been previously used to measure the stoichiometry of enzyme-aminoacyladenylate complex or the binding of amino acids or aminoacyl-tRNA to aminoacyl-tRNA synthetases (Kosakowski & Bóck, 1971;Hyafil et al, 1976; Güntner & Holler, 1979). Here this technique was applied to the determination of the kinetic rate constant for the dissociation of the phenylalanyladenylate-enzyme complex.…”

The binding of adenosine(5′)tetraphospho(5′)adenosine to calf thymus histones measured by non-equilibrium dialysis

“…The adduct of Ap4A with cis-diamminedichloroplatinum(II) has been prepared by the method of Segal et al (1984) and purified similarly to Ap4A (Pimmer et al, 1976). Non-equilibrium dialysis Dissociation constants and stoichiometries were measured by non-equilibrium dialysis as described previously (Colowick & Womack, 1969;Guntner & Holler, 1979;Muise & Holler, 1985). A dialysis apparatus was employed that consisted of a 1500 1dl (upper) reaction…”