The principal catalytic factor in the activation of tyrosine by the tyrosyl-tRNA synthetase is found to be improved binding of ATP in the transition state. The activation reaction involves the attack of the tyrosyl carboxylate on the .alpha.-phosphate group of ATP to generate a pentacoordinate transition state. Model building of this complex located a binding site for the .gamma.-phosphate group of ATP, consisting of hydrogen bonds with the side chains of Thr-40 and His-45. Removal of these groups by protein engineering shows that they contribute no binding energy with unreacted ATP but put all of their binding energy into stabilizing the [tyrosine .cntdot. ATP] transition state [the mutant tyrosoyl-tRNA synthetase(Thr-40 .fwdarw. Ala-40; His-45 .fwdarw. Gly-45) has the rate of formation of tyrosyl adenylate lowered by 3.2 .times. 105 but KS for ATP is lowered by only a factor of 5]. The side chains of these residues also provide a binding site for pyrophosphate in the reverse reaction. Thus, catalysis is accompanied by stabilization of the transition state by improved binding of a group on the substrate that is distant from the seat of reaction.

The naturally-occurring cyclic cystine-knot microprotein trypsin inhibitors MCoTI-I and MCoTI-II have been synthesised using both thia-zip native chemical ligation and a biomimetic strategy featuring chemoenzymatic cyclisation by an immobilised protease. Engineered analogues have been produced containing a range of substitutions at the P₁ position that redirect specificity towards alternative protease targets whilst retaining excellent to moderate affinity. Furthermore, we report an MCoTI analogue that is a selective low-µM inhibitor of foot-and-mouth-disease virus (FMDV) 3C protease, the first reported peptide-based inhibitor of this important viral enzyme.