The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: Enzymatic and comparative biophysical characterization

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulf olobus acidocaldarius (S-PPase) has been heterologously expressed in Escher ichia coli and could be purified in large quantities. S-PPase, previously d escribed as a tetrameric enzyme, was shown to be a homohexameric protein th at had catalytic activity with Mg2+ > Zn2+ > Co2+ much greater than Mn2+ mu ch greater than Ni2+, Ca2+, CD and FTIR spectra demonstrate a similar overa ll fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophi lus (T-PPase), The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model, S-PPase is extre mely heat resistant. Even at 95 degrees C the half-life of catalytic activi ty is 2.5 h, which is dramatically increased in the presence of divalent ca tions. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization, The T-m values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (cEDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochlorid e-induced unfolding follows an unknown mechanism with a biphasic kinetic an d an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and s how a sigmoidal and monophasic transition, indicating a change in global st ructure during unfolding, which can be described by a two-state process com prising dissociation and denaturation of the folded hexamer into six monome rs, The respective Delta G(N-->D)(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significant ly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ D elta Delta G(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S- PPase and T-PPase. (C) 1999 Academic Press.