Overexpression and Divalent Metal Binding Properties of the Methionyl Aminopeptidase from Pyrococcus furiosus

• Published in: Biochemistry (Easton) 2002-06, Vol.41 (23), p.7199-7208
• Main Authors: Meng, Lu, Ruebush, Shane, D'souza, Ventris M, Copik, Alicja J, Tsunasawa, Susumu, Holz, Richard C
• Format: Article
• Language: English
• Subjects: Aminopeptidases - biosynthesis; Aminopeptidases - chemistry; Aminopeptidases - genetics; Binding Sites; Cations, Divalent - analysis; Cations, Divalent - chemistry; Cobalt - analysis; Cobalt - chemistry; Enzyme Activation; Ferrous Compounds - analysis; Ferrous Compounds - chemistry; Glycine - chemistry; Hydrolysis; Kinetics; Metalloendopeptidases - biosynthesis; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metals - analysis; Metals - chemistry; Methionine - chemistry; Pyrococcus furiosus - enzymology; Pyrococcus furiosus - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Spectrophotometry, Ultraviolet; Substrate Specificity; Temperature; Zinc - analysis; Zinc - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi020138p

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 °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 ∼85 °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 °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 °C, indicating that the rate-li