{"created":"2023-06-20T13:20:12.489418+00:00","id":212,"links":{},"metadata":{"_buckets":{"deposit":"884f415e-7cd7-45d4-844c-2473eaf7d0fa"},"_deposit":{"created_by":1,"id":"212","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"212"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000212","sets":["2:427:9"]},"author_link":["7800","7799","7801"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"楊, 慧君"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ヤン, フォジャン"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2001-03-23"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"Part I   GENERAL INTRODUCTION\n  Myoglobin (Mb) functions as an oxygen storage and carrier protein in muscle.  This protein has been one of the most intensively investigated hemoproteins as evident from the accumulated biochemical and spectroscopic data.  It has protoporphyrin IX as a prosthetic group, and is the first protein structure determined to high resolution by X-ray crystallographic analyses.\n  Thus, myoglobin has ever been serving as a model system for the study of structure-function relationships in heme proteins.\n\nPart II  Chapter 1.  The Role of Val68 (E11) on Oxidation Activity and Enantioselectivity of Sperm Whale Myoglobin\n  To probe the role of the distal valine 68 (E11) in sperm whale myoglobin (Mb) on the oxidation activity, site-directed mutagenesis was performed.  A series of Mb mutants, H64D/V68X Mbs, have been prepared by replacing Val-68 with Gly, Ala, Ser, Leu, Ile, and Phe in H64D Mb.  All of the mutant proteins are stable enough to be purified except for the H640/V68G mutant.  The oxidation of the substrate thioanisole by H640/V68X Mb-I  besides H64D/V68S was monitored by stopped-flow spectrometer and the sulfoxidation rate constants increase in the order of Phe ≦ Val < Leu < Ala < Ile.  The results suggest that the volume of hydrophobic residue at the 68 position influences the sulfoxidation activity.  A similar pattern is observed for the catalytic sulfoxidation of thioanisole by H64D/V68X Mbs and H2O2.  The dominant product in the catalytic sulfoxidation is the R isomer for the H64D/V68A and H64D/V68S mutants, with more than 84% enantiomeric excess (% ee).  However, increasing the polarity of the distal pocket by substituting Ala-68 with Ser in H64D Mb decelerates the catalytic sulfoxidation rate by 2-fold.  On the other hand, the H64D/V681 mutant affords dominantly the S isomer with the highest turnover rate up to 413 turnover/min.  The substitution of Vat-68 with Leu has little effect on enantioselectivity in the catalytic sulfoxidation but increases the reactivity with H202.  Both the value of % ee and rate in the catalytic sulfoxidation decrease for H64D/V68F Mb in comparison with H64D/V68A Mb, implying a large benzyl side chain of phenylalanine at the 68 position inhibits the access of substrate to the heme pocket.  Furthermore, the crystal structure of the mutant, H64D/V68A, has confirmed the previous report (J. Am. Chem. Soc.  121, 9952 -9957, 1999, Matsui et at.) on catalytic mechanism and the spectroscopic studies on H64D/V68X Mb phenylethylamine complexes which are prepared to mimic the transition sale of thioanisole sulfoxidation, have provided some information on enantioselectivi1y in the sulfoxidation.\nChapter 2.  Conversion of Sperm Whale Myoglobin into a Catalase-like Enzyme\n  The sperm whale myoglobin active site mutants (F43H/H64A and F43H/H64N Mb) have been constructed to mimic the active site of catalase in which the distal histidine is suggested to facilitate compound I  formation with H2O2.  The F43H/H64A and F43H/H64N mutants exhibit 3.8- and 13-fold higher reactivity in the ABTS oxidation by H2O2 than the wild type, respectively.  However such mutation does not increase the reactivity of the ferric state with H2O2 enough to accumulate compound I  and even depress compound I  accumulation with mCPBA.  Some reasons for the failure in the observation of compound I  for the novel double mutants would be suggested when the crystal structural analysis of those mutants is completed.\n\nPart III Chapter 1.  Asymmetric Oxidation Catalyzed by Sperm Whale Myoglobin Mutants\n  The sperm whale myoglobin active site mutants (L29H/H64L and F43H/H64L Mb) have been shown to catalyze the asymmetric oxidation of sulfides and olefins.  Thioanisole, ethyl phenyl sulfide, and cis-β-methylstyrene are oxidized by L29H/H64L Mb with more than 95% enantiomeric excess (% ee).  On the other hand, the F43H/H64L mutant transforms trans-β-methylstyrene into trans-epoxide with 96% ee.  The dominant sulfoxide product in the incubation of alkyl phenyl thioethers is the R isomer;  however, the mutants afford dominantly the S isomer of aromatic bicyclic sulfoxides.  The results help us for the rationalization of the difference in the preferred stereochemistry of the Mb mutants-catalyzed reactions.  Furthermore, the Mb mutants exhibit the improvement of the oxidation rate up to 300-fold with respect to wild type.\nChapter 2.  Characterization of I107H/H64L Myoglobin Mutant\n  Since the alignment of the distal histidine is important for the reactivity with hydrogen peroxide as well as the prolonged life time of Mb-I  [0zaki et al. (1997), J. Am. Chem. Soc., 119, 6666], I107H/H64L Mb was constructed with an objective of obtaining highly active Mb in the reaction with H2O2.  However, the increased activities in peroxidation and peroxygenation are not observed for the I1O7H/H64L mutant in comparison with H64L and WT Mbs.  The finding indicates that not only the distance of distal histidine to the heme iron but also its conformation might be crucial in the activation of H2O2 for Mb.  In addition, attempts arc made to define the mechanism of influence of the distal histidine on regioselectivily in the coupled oxidation of several distal histidine relocation Mbs including I107H/H64L.  HPLC analysis of biliverdin isomers shows that relocation of the distal histidine at the 107 position (I107H/H64L Mb) affords the amount of γ-isomer to 22%, while L29H/H64L Mb almost exclusively gives γ-isomer compared with H64L and WT Mbs which mainly afford α-isomer.\n\nPart IV  SUMMARY AND CONCLUSION\n  In the present thesis, the author aimed to clarify enantioselectivity of oxidation reactions catalyzed by hemeproteins.  Sperm whale myoglobin (Mb) is employed as a model hemoprotein for the purpose, and some Mb mutants have been prepared by site-directed mutagenesis.\n  On the one hand, a series of H64D/V68X Mb mutants have been prepared to investigate the function of residues at the position 68 on the oxidation activity.  The results presented here demonstrate that the size and polarity of residues 68 in Mb play absolutely important roles on the oxidation activity and enantioselectivility in peroxidation and peroxygenation.  The changes in the oxidation activity would be rationalized by different reactivity of compound I  for H64D/V68X Mbs.  It has been clearly that rolarity of the active site instead of a general acid-base catalyst of residue Asp-64 is involved in calalytic mechanism.  Analysis of 1-phenylethylamine-Mb-complex crystal structure is under way in order to elucidate the precise substrate binding mode.  This type of information would provide a basis for the development of myoglobin mutants designed to catalyze enantioselective oxidation of interest, an important goal in synthetic organic chemistry.\n  On the other, substrate specificity is also important in controlling enatioselectivity of enzymatic oxidation.  Previously, L29H/H64L and F43H/H64L Mb were reported to exhibit catalytic turnover with high stereospecificity for the sulfoxidation of thioanisole and the epoxidation of styrene.  To determine how substrate structures influence on the enantioselectivity oxidations by Mb, the scope of asymmetric oxygenation by the use of various sulfides and styrene were explored for L29H/H64L and F43H/H64L Mb.  On the basis of changes in stereoselectivity, the substrate binding mode for Mb mutant-catalyzed reactions was rationalized.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第513号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"数物科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"07 構造分子科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2000"}]},"item_1_version_type_23":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"YANG, Hui Jun","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲513_要旨.pdf","filesize":[{"value":"333.4 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/212/files/甲513_要旨.pdf"},"version_id":"7d7843e2-7c65-4082-bb61-52d6b11002f7"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲513_本文.pdf","filesize":[{"value":"1.9 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文 / Thesis","url":"https://ir.soken.ac.jp/record/212/files/甲513_本文.pdf"},"version_id":"57783af5-422b-4156-b805-46c22484eb26"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"STEREOCHEMISTRY IN CATALYTIC OXIDATION BY HEME ENZYMES","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"STEREOCHEMISTRY IN CATALYTIC OXIDATION BY HEME ENZYMES"},{"subitem_title":"STEREOCHEMISTRY IN CATALYTIC OXIDATION BY HEME ENZYMES","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["9"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"212","relation_version_is_last":true,"title":["STEREOCHEMISTRY IN CATALYTIC OXIDATION BY HEME ENZYMES"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:59:09.539073+00:00"}