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Papers In Press, published online ahead of print November 30, 2000
Department of Biology, Okayama University, Okayama 700-8530
Corresponding Author: kimiyuki{at}cc.okayama-u.ac.jp
The CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, the over-expression in E. coli and the following one-step purification of spinach CtpA were established, in order to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on thylakoidal membrane. The successful accumulation of the functional CtpA in E. coli may argue against the possibility, based on the homology with E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, for example, by utilizing the ssrA-tagging system. The analysis using synthetic pD1 oligopeptide demonstrated that the enzymatic properties, including the substrate recognition, of the over-expressed CtpA with an extra sequence of GSHMLE at the N-terminus was indistinguishable from that of the authentic enzyme. The CtpA was insensitive to penem, which has been proved to inhibit some Ser/Lys type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e., the synthetic pD1 oligopeptide in solution and the pD1 in PSII-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and the affinity for the substrate, suggesting the presence of specific interaction of CtpA with factor(s) that modulates the pH dependency of proteolytic action in response to physiological conditions.
J. Biol. Chem, 10.1074/jbc.M008877200
Submitted on September 28, 2000
Revised on November 14, 2000
Accepted on November 30, 2000
Over-expression and characterization of carboxyl-terminal processing protease for precursor D1 protein: The regulation of enzyme-substrate interaction by molecular environments
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