Conformational changes tRNA-induced

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

One may arise by deprotonation of the reacting -NH2 group of the aminoacyl-tRNA (Eq. 29-1, step c). Conformational changes,387e which may be induced by proton movements, may also be encompassed within the array of pH-independent equilibria. 

Figure 29.30. Translocation Mechanism. In the GTP form, EF-G binds to the EF-Tu-binding site on the 50S subunit. This stimulates GTP hydrolysis, inducing a conformational change in EF-G, and driving the stem of EF-G into the A site on the 308 subunit. To accommodate this domain, the tRNAs and mRNA move through the ribosome by a distance corresponding to one codon.

This may however, be due to limitations In our model system using tBNA species with con lementary anticodons. On the other hand. It Is also possible that other components Involved In the rlbosomal decoding process as well as the proper codon are necessary to Induce a conformational change to allow the proper binding of the amlnoacyl-tRNA to the rlbosomal A-slte. 

At 50° C, enzyme-activated valine and isoleucine catalyzed the transfer of isoleucine to isoleucine tRNA, but failed to catalyze the transfer of valine to valine tRNA. As the temperature was increased from 50-80° C, the formation of isoleucyl tRNA decreased whereas that of valyl tRNA began to take place at 65° and increased up to 80° C. To explain these data, it was proposed that the temperature changes induced in tRNA conformational changes, which would either facilitate its reaction with the synthetase (in the case of valine) or interfere with it (in the case of isoleucine). 

The role of GTP in EF-Tu-promoted binding of aminoacyl-tRNA could be formulated as follows. First, a unique conformation of EF-Tu induced by GTP can select exclusively the aminoacylated form of tRNA in preference to its deacylated form. Second, the ternary complex, aminoacyl-tRNA-EF-Tu-GTP, is transferred to a precise location on the 508 ribosomal subunit through the conformation of EF-Tu-GTP favorable for interaction with ribosomes. The conformational change of aminoacyl-tRNA induced by complexing with EF-Tu-GTP may also serve for this interaction. Third, after the transfer of aminoacyl-tRNA to the A site of ribosomes, EF-Tu is to be released from ribosomes to reinitiate a new cycle of reactions. This could be accomplished by the hydrolysis of bound GTP to GDP. An additional advantage of the split of GTP is to shift the equilibrium irreversibly... 

There is little information about the details of the translocation mechanism. A continuous polyribonucleotide chain is not essential, as translocation can occur with individual trinucleotides. It seems likely that movement of the mRNA is dependent on and tightly coupled to that of the tRNA with the binding sites for the tRNA providing the precision for movement by exactly one codon. Presumably, binding of EF-G and GTP after release of EF-Tu-GDP following peptide bond synthesis induces a conformational change in the ribosome which leads to translocation. 

---