Formation of p-coumaric acid and o-coumaric acid from L-phenylalanine by microsomal membrane fractions from potato: Evidence of membrane-bound enzyme complexes.

The enzymes described here are the membrane-bound L-phenylalanine ammonia-lyase and cinnamate hydroxylase. Microsomes prepared from tubers of Solanum tuberosum L. are capable of converting L-phenylalanine into both o- and p-coumaric acid. Three microsomal fractions obtained by density gradient centrifugation were characterized by their equilibrium densities. Within various subfractions we found different patterns of distribution of the two enzymes, probably because of the extent of their fixed arrangement on a membrane area. Simultaneous incubation of the microsomal fraction with L-[4-ring-(3)H]-phenylalanine and trans-[3-(14)C]cinnamate indicated the existence of two pools of substrate available to cinnamate p-hydroxylase: cinnamate formed by the L-phenylalanine-ammonialyase reaction was a more effective substrate than cinnamate added to the incubation mixture. We conclude that the coumaric acids are formed from L-phenylalanine by a mechanism by which endogenously formed cinnamate is only partially equilibrated with exogenous cinnamate supplied in the incubation medium. This effect of enzyme cooperation is dependent on the integrity of membranes. The extent of cooperation was reduced by attempts to purify the microsomal membranes and by treatment in vivo with ethylene at high concentrations.