Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same K m and V max values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the V max value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e. binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.
