N-5,N-10-METHENYLTETRAHYDROMETHANOPTERIN CYCLOHYDROLASE FROM THE EXTREME THERMOPHILE METHANOPYRUS-KANDLERI - INCREASE OF CATALYTIC EFFICIENCY (K(CAT)/K(M)) AND THERMOSTABILITY IN THE PRESENCE OF SALTS

The activity of purified N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (I M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K(m) for N5,N10-methenyltetrahydromethanopterin from 2 mM to 40-mu-M and by increasing the V(max) from 2000 U/mg (k(cat) = 1385 s-1) to 13 300 U/mg (k(cat) = 9200 s-1). Besides increasing the catalytic efficiency (k(cat)/K(m)) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.