|Ph.D Student||Yifat Fundoiano-Hershcovit|
|Subject||Mechanistic and Structure-Function Studies on Bacterial|
|Department||Department of Biotechnology and Food Engineering||Supervisor||Full Professor Shoham Yuval|
|Full Thesis text|
Aminopeptidases (APs) are proteolytic enzymes that cleave the N-terminal amino acids from peptides and proteins. The overall goal of this research was to study the biochemical and catalytic properties of two bacterial aminopeptidaes from Bacillus subtilis (BSAP) and Streptomyces griseus (SGAP).
BSAP was cloned expressed in E. coli, purified and characterized. The enzyme is a metallopeptidase occupying two Zn molecules in its active site and exhibits high specificity towards basic amino acids.
Based on high-resolution crystal structures of SGAP, a catalytic mechanism was suggested involving Glu131 and Tyr246. To investigate the catalytic mechanism of SGAP, the gene was cloned and the protein was overexpressed in E. coli grown in the presence of 1M sorbitol to improve protein solubility. The E131A replacement in SGAP resulted in over 5-orders of magnitude decrease in kcat, but did not significantly affect Km. The two other replacements, E131Q and E131D, resulted in about 4-orders of magnitude reduction in kcat, consistent with Glu131 acting as a general-base. The catalytic constants (Km, kcat) in the temperature range of 293 to 329 K were determined for both SGAP and the E131D mutant. In both cases, the thermodynamic parameters for the reaction were similar for the binding step. In contrast, the E131D replacement resulted in an increase of 9 kJ mole-1 in the activation energy, emphasizing the crucial role of Glu131 in catalysis. The replacement of Tyr246 to Ser, Ala and Phe resulted in about two-orders of magnitude decrease in activity indicating that the phenolic hydroxyl of Tyr246 plays an important role in substrate hydrolysis. pH-Dependence activity profiles were measured and the value of enthalpy of ionization obtained, 30 ± 5 kJ mole-1, is typical for a zinc-bound water molecule. Fluoride acted as a pure non-competitive inhibitor of SGAP at pH values of 5.9 to 8. Since fluoride is likely to replace the bound water (pKa value of 7.0), the binding of the water/hydroxide molecule to both zinc ions is the same in the free enzyme as in the enzyme-substrate complex. Solvent isotope effect was measured at different pHs on SGAP and its E131D mutant. The results indicated that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. Overall the results support the proposed catalytic mechanism.