Mechanistic and structural studies of aminopeptidase from Streptomyces griseus and prolyl oligopeptidase from Pyrococcus furiosus

The first part of the dissertation illustrates that phosphate is a noncompetitive inhibitor for Streptomyces griseus aminopeptidase (sAP) between pH 5.85 and 9.0. The K_i of phosphate increased at higher pH values. A plot of pH versus pK _i reveals a pK_a of 7.05, likely due to ionization of H₂PO₄−. This noncompetitive inhibition pattern indicates that phosphate binding to sAP in solution is different from that proposed in the crystal structure, where phosphate was shown to bridges the two Zinc ions. Fluoride uncompetitively inhibits sAP from pH 5.5 to 9.0, with K_i being 3.72 ± 0.52 mM at pH 5.5 and 43.6 mM ± 0.91 at pH 9.0. A pK _a of approximately 6.2 was observed, likely due to a coordinated water. Paramagnetic effect on 31NMR relaxation of phosphate binding to CoZn-sAP indicates that only one phosphate is bound to sAP, which is at a minimum of 4.1 Å from the metal center. Along with chemical modification studies these results demonstrate that the phosphate-binding site is Arg-202 and that this residue plays an important role in the action of sAP.; The second part of the dissertation describes the catalytic mechanism of the prolyl oligopeptidase (POP) from the hyperthermophilic archaeon Pyrococcus furiosus (Pfu), which was investigated in both H₂O and D₂O toward the cleavage of Z-Ala-Pro- pNA and Z-Gly-Pro-pNA. The pH-rate profiles show a double sigmoidal pattern for the hydrolysis of Z-Gly-Pro pNA, which displayed pK_a's of 4.25 ± 0.31, 7.15 ± 0.13, and 9.07 ± 0.12. The temperature dependence of the p K_a values revealed a ΔH_ioniz of ∼4.7 kJ/mol for pK_es1 and ∼23 kJ/mol for p K_es2, suggesting that an aspartic acid deprotonation represents the first pK_a in the pH-rate profile and a imidazole deprotonation represents the second pK_a. A model has been constructed for Pfu POP based on crystal structure of porcine POP via sequence alignment, and is used for the discussion of the POP mechanism. Through site directed mutagenesis an active site tyrosine been changed independently to phenylalanine, showing a 42-fold decrease in k_cat suggesting that the role of Tyr-401 functions in substrate rearrangement.