SALT DEPENDENCE, KINETIC-PROPERTIES AND CATALYTIC MECHANISM OF N-FORMYLMETHANOFURAN - TETRAHYDROMETHANOPTERIN FORMYLTRANSFERASE FROM THE EXTREME THERMOPHILE METHANOPYRUS-KANDLERI - Research portal of Justus Liebig University Giessen:

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SALT DEPENDENCE, KINETIC-PROPERTIES AND CATALYTIC MECHANISM OF N-FORMYLMETHANOFURAN - TETRAHYDROMETHANOPTERIN FORMYLTRANSFERASE FROM THE EXTREME THERMOPHILE METHANOPYRUS-KANDLERI

Autorenliste: BREITUNG, J; BORNER, G; SCHOLZ, S; LINDER, D; STETTER, KO; THAUER, RK

Jahr der Veröffentlichung: 1992

Seiten: 971-981

Zeitschrift: European Journal of Biochemistry

Bandnummer: 210

Heftnummer: 3

ISSN: 0014-2956

Open Access Status: Bronze

DOI Link: https://doi.org/10.1111/j.1432-1033.1992.tb17502.x

Verlag: Wiley: No OnlineOpen

Abstract:

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) 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 revealed that phosphates and sulfates both affected the V(max) rather than the K. for CHO-MFR and H4MPT. At the optimal salt concentration and at 65-degrees-C, the V(max) was 2700 U/mg (I U = 1 mumol/min), the K(m) for CHO-MFR was 50 muM and the K(m) for H4MPT was 100 muM. At 90-degrees-C, the temperature optimum of the enzyme, the V(max) was about 2.5-fold higher than at 65-degrees-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-degrees-C decreased in the order K2HPO4 = (NH4)2SO4 much greater than KCl = NH4Cl = NaCl much greater than 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.

Zitierstile

Harvard-Zitierstil: BREITUNG, J., BORNER, G., SCHOLZ, S., LINDER, D., STETTER, K. and THAUER, R. (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), pp. 971-981. https://doi.org/10.1111/j.1432-1033.1992.tb17502.x

APA-Zitierstil: BREITUNG, J., BORNER, G., SCHOLZ, S., LINDER, D., STETTER, K., & THAUER, R. (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. https://doi.org/10.1111/j.1432-1033.1992.tb17502.x

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