Studies on Carboxyl Proteinases : Comparative Analysis of Functional Structures and Catalytic Mechanisms of Proctases A and B

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1.By specific chemical modifications, carboxyl groups were shoun to be involved in the active site of Proctase A as in the case of Proctase B.Asp14 and Asp 111 in the heavyc hain of Proctase A were indicated to be the catalytic groups by site-directed mutagenesis studies using an E.coli expression system for pro-Proctase A.2.A cDNA of prepro-Proctase B was cloned and sequenced, giving the complete amino acid sequence of the preproenzyme. Pro-Proctase B was shown to be autocatalytically activated under acidic conditions. Site-direcited mutagenesis studies on pro-Proctase B revealed that Lys 36 and Arg 32 are important for the shroctural stability of the proform and its activation, respectively.3.The expression system of pro-Proctase A in E.coli was established, and the proenzyme was shown to be autocatalytically activated under acidic conditions like pro-Proctase B.4.The profiles of denaturation of Proctase A at different pHs and temperatures were in vestigated using various physico-chemical methods in clading NMR and circular dichroism, which revealed unique denaturation profile of the enzyine.5.Nearly all proton signals in NMR of the light chain of Proctase A were assigned and the three-dcmensional structure of the chain was determined. Further, a method for preparation of isotopically labeled Proctase A using Aspergillus niger was established, and using these ^<15>N and/or ^<13>C-labeled Protase A,the three-dimensional structure was investigated.6.A unique substrate specificity of Proctase A was elucidatid using oxidized B chain of insulin ets, which in as quite differant from that of Proctase B.In addition, the interaction of an inhibitor peptide with Proctase A was investigated by NMR.7.Isomorphous metal (Pt and Hg)derivatives of Proctase A were crystallized and the three-dimensional shructure of the enzyrne was partly elarified by X-ray crystallographic analysis.