Salt dependence, kinetic properties and catalytic mechanism of N‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri
10.1111/j.1432-1033.1992.tb17502.x
Crossref journal-article
Wiley
European Journal of Biochemistry (311)
Abstract

N‐Formylmethanofuran(CHO‐MFR): tetrahydromethanopterin(H4MPT) formyltransferase (for‐myltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100‐fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N‐terminal amino acid sequence of the polypeptide was determined.The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half‐maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revcaled that phosphates and sulfates both affected the Vmax rather than the Km for CHO‐MFR and H4MPT. At the optimal salt concentration and at 65°C, the Vmax was 2700 U/mg (1 U = 1 μmol/min), the Km for CHO‐MFR was 50 μM and the Km for H4MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the Vmax was about 2.5‐fold higher than at 65°C.Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90°C decreased in the order K2HPO4= (NH4)2SO4≫ KCI = NH4Cl = NaCl ≫ Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary‐complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.

Bibliography

BREITUNG, J., BÖRNER, G., SCHOLZ, S., LINDER, D., STETTER, K. O., & THAUER, R. K. (1992). Salt dependence, kinetic properties and catalytic mechanism of N‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri. European Journal of Biochemistry, 210(3), 971–981. Portico.

Authors 6
  1. Jürgen BREITUNG (first)
  2. Gerhard BÖRNER (additional)
  3. Sabine SCHOLZ (additional)
  4. Dietmar LINDER (additional)
  5. Karl O. STETTER (additional)
  6. Rudolf K. THAUER (additional)
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Dates
Type When
Created 20 years, 6 months ago (March 4, 2005, 2:52 a.m.)
Deposited 1 year, 11 months ago (Oct. 1, 2023, 4:52 a.m.)
Indexed 3 months, 2 weeks ago (May 21, 2025, 6:04 p.m.)
Issued 32 years, 9 months ago (Dec. 1, 1992)
Published 32 years, 9 months ago (Dec. 1, 1992)
Published Online 20 years, 6 months ago (March 3, 2005)
Published Print 32 years, 9 months ago (Dec. 1, 1992)
Funders 0

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@article{BREITUNG_1992, title={Salt dependence, kinetic properties and catalytic mechanism of N‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri}, volume={210}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1992.tb17502.x}, DOI={10.1111/j.1432-1033.1992.tb17502.x}, number={3}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={BREITUNG, Jürgen and BÖRNER, Gerhard and SCHOLZ, Sabine and LINDER, Dietmar and STETTER, Karl O. and THAUER, Rudolf K.}, year={1992}, month=dec, pages={971–981} }