The Extreme Thermostable Pyrophosphatase fromSulfolobus acidocaldarius:Enzymatic and Comparative Biophysical Characterization

Recombinant pyrophosphatase from the hyperthermophilic archaebacteriumSulfolobus acidocaldarius(S-PPase) has been heterologously expressed inEscherichia coliand could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+> Zn2+> Co2+⪢ Mn2+⪢ Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases fromE. coli(E-PPase) andThermus thermophilus(T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95°C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+per monomer is needed for catalysis, but no more than one Mg2+per monomer is sufficient for thermal stabilization. TheTmvalues for S-PPase are 89°C (+EDTA), 99°C (+Mg2+), and >100°C (+Mn2+), compared to 58°C (+EDTA), 84°C (+Mg2+), and 93°C (+Mn2+) for E-PPase and 86°C (+EDTA), 99°C (+Mg2+), and 96°C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective ΔGN→D(25°C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ΔΔG(25°C) is 16 kJ/mol for E-PPase and 5.5–8 kJ/mol for S-PPase and T-PPase.