Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PjMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PjMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(H). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P I position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest Km values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (k(cat)/K-m) of PfMetAP-II for MP-p-NA at 30 degreesC was 799 500 and 340 930 M-1 s(-1) for Co(II)- and Fe(H)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with I equiv of Co(II) or Fe(II), and the dissociation constants (K-d) for the first metal binding site were found to be 50 +/- 15 and 20 +/- 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 +/- 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(H) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be similar to85 degreesC at pH 7.5 in 25 MM Hepes and 150 mM KO buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degreesC at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and Km values increased with increasing temperature. An Arrhenius plot was constructed from the k(cat) values and was found to be linear over the temperature range 25-85 degreesC, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(H)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 degreesC are as follows: DeltaGdouble dagger = 46.23 kJ/mol, DeltaHdouble dagger = 10.79 kJ/mol, and DeltaSdouble dagger = -119.72 J.mol(-1).K-1 for Co(II)-loaded PfMetAP; DeltaGdouble dagger = 46.44 kJ/mol, DeltaHdouble dagger = 16.94 kJ/mol, and DeltaSdouble dagger = -99.67 J.mol(-1).K-1 for Fe(H)-loaded PfMetAP. Interestingly, at higher temperatures (> 50 degreesC), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degreesC) than Co(II)-loaded PfMetAP-II.