Salt dependence, kinetic properties and catalytic mechanism of N‐formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri

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 V_max rather than the K_m for CHO‐MFR and H₄MPT. At the optimal salt concentration and at 65°C, the V_max was 2700 U/mg (1 U = 1 μmol/min), the K_m for CHO‐MFR was 50 μM and the K_m for H₄MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the V_max 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.