Affinity labelling of tryptophanyl-transfer RNA synthetase

Abstract

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme. Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α 2 An ATP analogue, γ-( p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K 1 = 1 × 10 −3m ( K mfor ATP = 2 × 10 −4m). The covalent photoaddition of azido-ATP ‡ ‡ Abbriviations used: azido-ATP, γ-( p-azidoanilide)adenosine triphosphate; TRSase, tryptophanyl-tRNA synthetase (EC 6.1.1.2); R-Trp-tRNA, a residue of ambucilyl-tryptophanyl-tRNA covalently bound to the protein; Cl-R-Trp-tRNA, N-chlorambucilyl-tryptophanyl-tRNA; chlorambucil, γ- p-bis(2-chloroethyl)-aminophenyl-butyrie acid; -R-tryptophan, a residue of ambucilyl-tryptophan covalently bound to the protein; BDC, benzoylated diethylaminoethyl cellulose. results in complete loss of enzymatic activity in both the ATP-[ 32P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme. Two binding sites for tRNA Trp have been found from complex formation at pH 5.8 in the presence of Mg 2+. The two tRNA molecules bind, with K dis = 3.6 × 10 −8m and K dis = 0.9 × 10 −6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA. N-chlorambucilyl-tryptophanyl-tRNA Trp and TRSase form a complex with K dis = 5.5 × 10 −8m at pH 5.8 in the presence of 10 m m-Mg 2+. This value is similar to the value of K dis for tryptophanyl-tRNA of 4.8 × 10 −8 m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA Trp whereas the tRNA devoid of tRNA Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected. Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [ 32P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[ 32P]pyrophosphate exchange and aminoacylation activity. The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein. Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.