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The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization.
Arch Biochem Biophys 1999; 363(1):135-47AB

Abstract

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and 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 from E. coli (E-PPase) and Thermus 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 degrees 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. The Tm 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 (+EDTA), 99 degrees C (+Mg2+), and 96 degrees 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 DeltaGN-->D(25 degrees 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+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.

Authors+Show Affiliations

Institute of Biochemistry, Medical University of Lübeck, Ratzeburger Allee 160, Lübeck, D-23538, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10049508

Citation

Hansen, T, et al. "The Extreme Thermostable Pyrophosphatase From Sulfolobus Acidocaldarius: Enzymatic and Comparative Biophysical Characterization." Archives of Biochemistry and Biophysics, vol. 363, no. 1, 1999, pp. 135-47.
Hansen T, Urbanke C, Leppänen VM, et al. The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. Arch Biochem Biophys. 1999;363(1):135-47.
Hansen, T., Urbanke, C., Leppänen, V. M., Goldman, A., Brandenburg, K., & Schäfer, G. (1999). The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. Archives of Biochemistry and Biophysics, 363(1), pp. 135-47.
Hansen T, et al. The Extreme Thermostable Pyrophosphatase From Sulfolobus Acidocaldarius: Enzymatic and Comparative Biophysical Characterization. Arch Biochem Biophys. 1999 Mar 1;363(1):135-47. PubMed PMID: 10049508.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization. AU - Hansen,T, AU - Urbanke,C, AU - Leppänen,V M, AU - Goldman,A, AU - Brandenburg,K, AU - Schäfer,G, PY - 1999/3/2/pubmed PY - 1999/3/2/medline PY - 1999/3/2/entrez SP - 135 EP - 47 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 363 IS - 1 N2 - Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and 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 from E. coli (E-PPase) and Thermus 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 degrees 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. The Tm 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 (+EDTA), 99 degrees C (+Mg2+), and 96 degrees 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 DeltaGN-->D(25 degrees 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+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/10049508/The_extreme_thermostable_pyrophosphatase_from_Sulfolobus_acidocaldarius:_enzymatic_and_comparative_biophysical_characterization_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(98)91072-4 DB - PRIME DP - Unbound Medicine ER -