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

N ‐Formylmethanofuran(CHO‐MFR): tetrahydromethanopterin(H 4 MPT) 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 K 2 HPO 4 > (NH 4 ) 2 SO 4 > K 2 SO 4 > Na 2 SO 4 > Na 2 HPO 4 . The salts KCl, NaCl and NH 4 Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half‐maximal activity, 1 M K 2 HPO 4 and 1.2 M (NH 4 ) 2 SO 4 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 H 4 MPT. 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 H 4 MPT 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 K 2 HPO 4 = (NH 4 ) 2 SO 4 ≫ KCI = NH 4 Cl = NaCl ≫ Na 2 SO 4 > Na 2 HPO 4 . 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 .