The mechanism of substrate recognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by x-ray crystallography and site-directed mutagenesis

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal py roglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a tr ansition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the in hibitor (71:O . . .H-N:Inh and Gln71:N-H . . . OE:Inh and the pyrrolidone r ing of the inhibitor was inserted into the hydrophobic pocket composed of P he-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143, To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two ph enylalanine mutants F13Y and F142Y did not exhibit significant changes in k inetic parameters compared with the wild-type enzyme, The catalytic efficie ncies (K-cat/K-m) for the F13A and F142A mutants were less than 1000-fold t hat of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophob ic pocket compared with the wild type. These findings indicate that the mol ecular recognition of pyroglutamic acid is achieved through two hydrogen bo nds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is mo re important to the hydrophobic interaction than is Phe-142, and furthermor e Phe-13 serves as an "induced fit" mechanism.