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Abstract
The kinetics of carboxylic acid methylation by chloromethane (CH3Cl) in mycelia of the fungus Phellinus pomaceus were examined. Substantial incorporation of C2H3-into ester was observed within 5 min of addition of C2H3Cl to washed mycelia in the presence of the non-physiological acceptor butyric acid, rendering it unlikely that CH3Cl was converted to a diffusible intermediate before acting as methyl donor. The rate of methyl butyrate biosynthesis attained a maximum of 0·14 mol g−1 h−1 at 1·5 mm-butyric acid, with higher concentrations causing increasing inhibition. Exogenous CH3Cl did not affect methyl butyrate production implying that the rate of CH3Cl biosynthesis did not limit methylation. However, C2H3,-incorporation from exogenous C2H3Cl into methyl butyrate rose sharply from 20 to 60% between 15 and 4 mm-butyric acid, suggesting inhibition of CH3Cl biosynthesis by the acid, an interpretation supported by the rapid decline in gaseous CH3Cl release by mycelia between 1·5 and 2 mm-butyric acid. With the natural acceptor benzoic acid as substrate a significant increase in the rate of ester biosynthesis was obtained in the presence of exogenous CH3Cl. Ester biosynthesis was maximal (0·18 μmol g−1 h−1) at 0·5 mm-benzoic acid but fell extremely rapidly with increasing concentration. As with butyric acid supraoptimal concentrations halted CH3Cl release and increased C2H3-incorporation from exogenous C2H3Cl. Studies on C2H3-incorporation from exogenous C2H3Cl into ester revealed a linear relationship between the logarithm of the percentage C2H3-incorporation and the logarithm of C2H3Cl concentration with both butyric and benzoic acids as substrate, suggesting that exogenous C2H3Cl competed with endogenously synthesized CH3Cl for adsorption at a solid interface, possibly a membrane within the cell, prior to reaction of the compound at the active site. When mycelia were grown in the presence of different halide ions, greater methylating activity was found in Br−- and I−-grown mycelia. The system had a higher affinity for CH3Br and CH3I as methyl donors than CH3Cl. Fluoromethane was not a substrate for the methylating system nor did it act as a competitive inhibitor.
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