Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase.

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.