Abstract
In order to analyze the structural requirement(s) for proteolytic cleavage, synthetic oligopeptides corresponding to the carboxyl-terminal (COOH-terminal) sequence of the precursor to the D1 protein (pD1) of the photosystem II reaction center, with or without substituted side chain(s) around the cleavage site, were subjected to enzymatic analysis with partially purified processing protease from spinach. The efficiency of action as a competitive inhibitor of the enzymatic cleavage of the COOH-terminal extension, as well as the capacity to serve as a substrate, was used as an indication of effective binding to the protease. Neither a COOH-terminal fragment consisting of the 9 amino acids that are cleaved from pD1 by the protease nor a COOH-terminal fragment of the mature protein consisting of 15 amino acids inhibited the enzymatic processing of pD1. By contrast, a COOH-terminal fragment of pD1 consisting of 24 amino acids, which included the sequences of both the COOH-terminal extension and the COOH-terminal 15 amino acids of the mature protein, was effective both as a competitive inhibitor and as a substrate. This result suggests that the structure formed by linkage between these two parts of the protein moiety is important in the substrate-enzyme interaction. Among substitutions around the cleavage site, the replacement of Leu-343 by Ala (L343A) specifically destroyed the ability of the oligopeptide to serve as either a substrate or an inhibitor, suggesting that the presence of the hydrophobic Leu residue is crucial for the formation of the recognition site. A series of six substitutions at Ala-345 had marked effects on the value of Vmax, without affecting the binding affinity, as represented by Km; the order of substitutions at residue 345 in terms of their effects on Vmax was Ala,Ser,Phe,Cys > Gly > Val >> Pro. With a Pro residue at position 345, the oligopeptide was practically inactive as a substrate.
Highlights
From the +Department of Biology, Okayama University, Okayama 700, Japan and the §Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Okazaki 444, Japan
In order to analyze the structural requirement(s) for proteolytic cleavage, synthetic oligopeptides corresponding to the carboxyl-terminal (COOH-terminal) sequence of the precursor to the Dl protein of the photosystem II reaction center, with or without substituted side chain(s) around the cleavage site, were subjected to enzymatic analysis with partially purified processing protease from spinach
Inhibitory Effects of COOH-terminal Oligopeptides-As described in the previous study [28], synthetic oligopeptides corresponding to the deduced COOH-terminal sequence of pD1 of spinach (Fig. 1), which can be used as the substrate for the isolated COOH-terminal processing protease, have an inhibitory effect on the enzymatic reaction with in vitro transcribed and translated full-length pD1 as substrate
Summary
The efficiency of action as a competitive inhibitor ofthe enzymatic cleavage of the COOH-terminal extension, as well as the capacity to serve as a substrate, was used as an indication of effective binding to the protease. An unusual property of this essential subunit of the PSII reaction center is, the extraordinarily high rate of its turnover in vivo [6, 7], which is thought to represent the damage-repair cycle of PSII [8] In this metabolic cycle, the Dl subunit is synthesized by thylakoid-bound ribosomes on the stromal surface of membranes as a precursor protein, which has a short carboxyl-terminal (COOH-terminal) extension [9, 10].
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