Biochemical and NMR studies of the reaction mechanisms of PBG deaminase and papain /
| Main Author: | |
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1989.
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| Subjects: | |
| Online Access: | ProQuest, Abstract Link to OAKTrust copy |
| Abstract: | The properties and covalent complexes of phorphobilinogen (PBG) deaminase and papain were studied by 13C NMR spectroscopy and traditional biochemical techniques in order to develop mechanistic rationales for these enzymes. PBG deaminase was overexpressed from a recombinant strain Escherichia coli (E. coli) containing the plasmid pBG101 with the hemC gene. The enzyme was isolated and purified to a single band of about 34,000 daltons on SDS polyacrylamide gel electrophoresis. The physical and catalytic properties of the E. coli enzyme were examined and compared to PBG deaminase from other sources. Of particular interest was the discovery of active isozymes which differed by their net charge and were separable on nondenaturing electrophoresis. The active site of E. coli PBG deaminase was found to contain a novel cofactor which had previously been undetected. The structure of the cofactor was assigned by 13C NMR to a dipyrromethane (DPM) derivative covalently and permanently bound to the enzyme. A hemB- strain of E. coli produced apoenzyme which was inactive. The substrate analog 2-bromo-[2,11-13C] PBG was determined to be a suicide-substrate inhibitor which covalently added to the DPM. Stable enzyme-substrate complexes corresponding to ES1, ES2, and ES3 were produced and separated on nondenaturing electrophoresis. The cofactor was found to be anchored within the active site to the sulfhydryl group of Cys-242 by cleavage experiments and site-specific mutagenesis. An overall mechanism was proposed which indicated the DPM cofactor was the site of head-to-tail tetramerization of PBG to hydroxymethylbilane. Papain was reversibly inactivated by N-Ac-L- and N-Ac-D-Phe-[1-13C]glycinal with Ki values of 2.6 x 10^-8 M^-1 and 13.3 x 10^-8 M^-1, respectively. The formation of diastereomeric thiohemiacetals with the 13C-enriched aldehydes was detected by 13C NMR spectroscopy. An unusually large downfield chemical shift was observed for the papain-ketone complexes with CBZ-Lys-[2-13C]CMK, CBZ-Gly-[2-13C]CMK and bromo-[2-13C]acetone. Based upon the 13C NMR data from these enzyme-inhibitor complexes a possible strain mechanism and the stereospecificity of the S1 and S2 binding sites were discussed with respect to papain catalyzed hydrolysis. |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Chemistry." |
| Physical Description: | xii, 194 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |