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 PfMetAP-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 PfMetAP-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(II). 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 P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest K(m) 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 degrees C was 799 500 and 340 930 M(-1) s(-1) for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 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(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be approximately 85 degrees C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and K(m) 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 degrees C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 degrees C are as follows: DeltaG++ = 46.23 kJ/mol, DeltaH++ = 10.79 kJ/mol, and DeltaS++ = -119.72 J.mol(-1).K(-1) for Co(II)-loaded PfMetAP; DeltaG++ = 46.44 kJ/mol, DeltaH++ = 16.94 kJ/mol, and DeltaS++ = -99.67 J.mol(-1).K(-1) for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (> 50 degrees C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 degrees C) than Co(II)-loaded PfMetAP-II.
Meng
Lu
L
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA.
Ruebush
Shane
S
D'souza
Ventris M
VM
Copik
Alicja J
AJ
Tsunasawa
Susumu
S
Holz
Richard C
RC
D000626:Aminopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002413:Cations, Divalent / Q000032:analysis / Q000737:chemistry; D003035:Cobalt / Q000032:analysis / Q000737:chemistry; D004789:Enzyme Activation; D005296:Ferrous Compounds / Q000032:analysis / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008670:Metals / Q000032:analysis / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000032:analysis / Q000737:chemistry
D002413:Cations, Divalent; D005296:Ferrous Compounds; D008670:Metals; D011994:Recombinant Proteins; D003035:Cobalt; D008715:Methionine; D000626:Aminopeptidases; C106541:methionine aminopeptidase 2; D008666:Metalloendopeptidases; D015032:Zinc; D005998:Glycine