The kinetics of phenol methylation by chloromethane (CHCI) in intact mycelia of the fungus were examined. Anisole was produced linearly with respect to time at a rate of 6.3 nmol g h over 16 h when washed mycelia were incubated with phenol and CHCI. Incorporation of CH- label into anisole attained a plateau value of 27% within 2 h. The rate of anisole production under N was only 36% of that in air, and the proportion of CH- label incorporated from exogenous CH- increased from 30 to 45% under these conditions. The rate of methylation attained a maximum in 10 mM-phenol, but the level of activity was only about 10% of that of the fungal carboxyl-methylating system. The presence of exogenous CHCI clearly stimulated anisole formation, demonstrating that the rate of CHCI biosynthesis limited methylation to some extent. In contrast to the carboxyl-methylating system, no significant inhibition of methylation was observed at methyl acceptor concentrations up to 17.5 mM, and no sharp fall in the rate of gaseous CHCI release or rapid increase in CH-incorporation were observed at supraoptimal concentrations of acceptor, indicating that endogenous CHCI biosynthesis was not inhibited under these conditions. Investigations of the rate of CH- incorporation into anisole from exogenous CHCI showed a linear relationship between the logarithm of % CH- incorporation and the logarithm of CHCI concentration, suggesting that, as postulated for the carboxyl-methylating system, the CHCI-synthesizing and CHCI-utilizing enzyme systems were situated on either side of a membrane within the cell.


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