関西学院大学リポジトリ

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(1)Stmct皿m1S伽d．ies om皿mzymaticReactiom of肋。teria1CopPerAmi皿e Oxi曲se. A1）octom1Thesis by. MISUMIK岨AOKへ. S㎜bmitte砒。仙e G醐伽ate Schoo1ofSciem㏄a皿dT㏄hmo1ogy Kwamsei Gak皿im Umiversity Japam. Jam㎜3ry，2011.

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(3) ACκnOW〃e的me耐S This is a thesis的r doctora1degree of∫oゴemoe，submitted to Kwansei Ga㎞in University．. The study in this thesis has be㎝per的㎜ed under the direction ofPro危ssor Hiroshi. Yamaguchi in the Depaれment of Chemistry，Schoo1of Science and Techno1ogy，Kwansei. Ga㎞in University丘。m2005t02011． His hear脆1t appreciation goes to Pro￡Hiroshi Yamaguchi的r his giving the author a. chance to study on this fie1d，cease1ess guidance，worthy suggestions and encouragement throughout this study．The author wou1d−1ike to express his sincerest gratitude to a冊1iate Pro￡. Takashi Kumasaka in Japan S〉mchrotron Radiation Research㎞stitute br his genia11eading，. he1p制suggestion，fmit冊1suppo血and−encouragement．Grateh1ac㎞ow1edgement is dedicated to Prof−Katsuyuki Tanizawa and associate Pro￡Toshihide Ok則’ima in the Institute ofScienti丘。. and Industria1Research，0saka University for kind gαidance，simu1ation discussions and he1p血1 SuggeStiOnS．. The author ac㎞ow1edges to Dr．“shikazu Kawano，Dr．Takaaki Hikima，Dr Tsuyoshi. Ueno andProf．MasakiYamamoto inR皿ENHarimaInstituteandDr．SeikiBaba，Dr．Nobuhiro Mizuno，DL Nobutaka Shimizu in Japan Synchrotron Radiation Research Institute，and Pro￡. Atsushi Nakagawa，Assistant Pro￡Eiki Yamashita and Dr．Masato Ybshimura，in Osaka University for woれhy suggestions and suppoれin the sing1e−cワsta1microspectrophotometer measurement and X−ray di附action experiments． The author is a1so grate血1to Dn Keiichi Hosokawa in Institute ofBio−Microbio1ogy for. his hea11二y suggestion．The author is thank血1 Dr． Tatsuya Uchida in TAISHO PHAKMACEUTICAL C0．，LTD．br his use冊1he1p．He ac㎞ow1edges Dr Len Ito in Japan Synchrotron Radiation Research Institute and Dr．Takeshi Hiromoto in Nagoya City University for their support in X−ray crysta11ographic ana1ysis．The author sincere1y thanks Dr Seiichiro. Kishishita inNovozymes Japan Ltd．br his he1p肋1discussion and woihy suggestion．. It shou1d be emphasized that the contributions of Mr．MasaWki Otsu，Ms．AWko Tominaga，Ms．Hiroko Oya，who participated in some ofthe work presented here，are great1y iii.

(4) apPreciated．He wou1d1ike to thank Ms．Satomi Uramoto，Mr．Masaki0㎞mura，Mr・Ryosuke Satoh in Ki吐i University，Mr．Keisuke Yokoya and Mr．Naonori Kagota br suppoれhis 1aboratory1ifさ．. The author is a1so thank血1Pro￡Yasushi Kai in Fu㎞i University of Techno1ogy，Pro￡. Y功i Hidaka in Kinki University，Pro￡Reiko Sugiura in Kinki University，Pro￡Noritake Yasuoka in University ofHyogo，Pro￡Shinichi Matsuki，Prof．Tadaaki Kaneko，Prof．Atsus㎞. Kurita，Pro￡Y血kihiro Ozaki，Prof．Shinsuke Fujiwara，Ms．Noriko Fu㎞da and Mr．Kensuke Nakajima的r unceasing suppoれduring his work and university1ife．. Theauthorcamotfbrgettoexpresshisgreattha欣stoa11theco11eagues，pastandpresent， in the Prof．Yamaguchi’s1aboratory，the Prof，Tanizawa’s1aboratory，the a脆1iate Prof．. Kumasaka’s1aboratory and the Pro￡地mamoto’s1aboratory．Without their support and encouragemcnt，he wou1d not have gottenthroughtheperiod concentrating onthis research． Fina11y，the author wou1d1ike to express his deep appreciation to his曲mi1y，Shigeki，. Tadako and Noriyoshi for their un勉i1ing understanding and encouragement throughout the period．. Depa廿ment ofChemistry. Graduate Schoo1of Science and Tech皿。1ogy Kwansei Ga㎞in University Japan. January，2011. iV.

(5) Coηね耐s Ackmw1edgememts. iii. Comtemts. V. AmimAcids. Viii. List ofAbbmviatiom. GemmHmtmd11ctiom. Seωom I. ix. 1. I．1Pre曲。e. I．2Pheny1ethy1amine Oxidase丘。mル。加。ゐααe7gZoわ伽7〃∫（AGA0）. I．3Structura1Studies on Enzymatic Reaction ofAGA0. Sectiom II. Experim㎝ta1Pm㏄d㎜res. 9. II．1Puri丘。ation. II．1．10verexpression ofApo−AGAO II．1．2Puriication ofHo1o−AGAO II．2Preparation ofCrysta1s II．2．1Crysta11ization ofHo1o−AGAO. II．2．2TrappingReactionIntemediatesofReductiveHa1トreaction II．2．3Xenon Comp1exation. II．3UV／VisAbso印tionMeasurements ofCrysta1s II．3．1Sing1e−Crysta1Microspectrophotometry. V.

(6) II．4X−ray Di耐action Experiments. II．4．1X−rayDi航actionMeasurement and Stmcture Re丘nement. Sectiom III. UV〃is Abso叩tio皿Spectm of Crys伽1s d㎜ri皿g C3伽1ytic Reactiom. 19. III．1Sing1e−crysta1Microspectroscopy. Sectio皿IV. Stn1ct11m1C11amges ofRed㎜cdve Ha昨Re3ctiom. 23. 1V．1StmctureDete㎜inations IV．1．1Ho1o−AGAO Stmcture. lV．1．2Michae1is Comp1exofAGAO andPEA rゾ．1．3Substrate and Product Schifr−bases. IV．1．4AGA0／HY1Comp1ex（Comp1ex ofReducedAGAO and Product）. SectiomV. Dioxygem−bimdi皿g sites by Usimg Xe Comp1ex. 39. V1Xe−AGA0／HY1Comp1ex. Sectiom VI. Disc皿ssioms. VI．1Reductive Ha1トreaction. VI．1．1ReactionMechanismofWTAGAOwithPEA VI．2Di脆rence ofReaction Rate between Each Subunit. VI．2．1Stmctura1Change ofAm1I VI．2．2Stmctura1Di脆rence inActive Site ofRTO. VI．2．3Mo1ecu1arPackingofRTO inCrysta1 Vi. 47.

(7) VI．3Initia1Step ofOxidative Ha1f−reaction. VI．3．1PathwayofProvidedMo1ecu1arOxygen. Comc111sioms. 64. Referemes. 66. List of P㎜b1ic3tioms. 70. Papers. Reports. List of Presemtatioms. 73. ㎞tematiOna1 Domestic. Awards. 79. Vii.

(8) Am7nO A07dS. ←. ト A1a血me 刈a（A）. A㎎阯m. A5岬mgi11o. A5m㈹. Arg岬）. AΨ3市ic8dd A5p（D）. Cy前8im Cis（C）. ㌔㌔ G111伽miI1e G1I1（Q）. Glut3皿ic肌id G1山（E）. G・1ycim G1y（G）. 正皿一tidi11e. 肋岬. ISO1e㎜Cim8. 皿e（1）. ㌔ Le凹。血e. 比皿①）. Lysim Ly5①9. M舳i㎝im Ph㎝y13hmi11o Pmmm Mot⑭④. pho㈹. 十. ト十. V111. pm四）. Ser血io. ■hl・eOmime. T㎜t0両㎜. Tyrosim6. Sor（S）. 1h（D. ■叩⑭η. 11W OO. Va1i皿6. Va1Cり.

(9) 1X.

(10) L7S‘0fAbb”e〃a市0nS ABTS. 2，2，一azinobis（3−ethy1benzothiazo1ine−6−su1ibnic acid）. AGAO. ル。加。わααe7970わ狗舳ゴ∫pheny1ethy1amine oxidase. Amp. ampici1in. BSAO. bovine semm amine oxidase. CTQ. cysteine t1＝yptophy1quinone. DDC. diethy1dithiocarbamic acid. DEAE. （d．iethy1aminoethy1）ce11u1ose. DPQ. dopaquinone. DTT. dithiothreito1. ECAO. ふ。加7た〃αcoZゴtyramine oxidase. EDTA. ethy1enediaminetetraacetic acid. FAD. F1avinAdenine Dimc1eotide. FMN. FravinMononuc1eotide. FPLC. Fast Protein Liquid Chromatograph. HEPES. 2一［4一（2−Hydroxyethy1）一1−pipcraziny1］ethanesu1曲nic acid. HPAO. 肋〃∫e〃〃αρoケmoψ伽methy1amine oxidase. HY1. pheny1aceta1dehyde. IPTG. isopropy1一β一D−thioga1actopyranoside. LTQ. 1ySy1tyrOSy1quinOne. NMR. nuC1earmagnetiC reSOnanCe. OD. optica1density. PAGE. po1yacry1amid ge1e1ectrophoresis. PDB. Protein Data Bank. PEA. 2−pheny1ethy1amine. PMSF. pheny1methy1su1わny1f1uo㎡de. PPLO. Pだみゴαρo∫coガs1ysi1oxidase. X.

(11) pQQ. pyrO10quinO1ine quinOne. PSAO. 〃舳mα∫αcゴwm diamine oxi（1ase. RMSD. r00t−mean−square＿deviation. SDS. sodium dodecy1su1曲te. SDS−PAGE. sodium dodecy1su1fatepo1yacワ1amide ge1e1ectrophoresis. SP㎡ng−8. Super Photon ring−8GeV. TPQ. 2，4，5−trihydroxypheny1a1any1quinone. TPQ。、. topaquinone（TPQintheoxidizedfom）. TPQ。、b. substrate Schi任＿base. TPQp。・. product Schifr−base. TPQ、。d. amonoresorcino1（TPQinthereducedfom）. TPQ。。. topasemiquinone. TPQim．. iminOquinOnOe. TTQ. tryptophan tryptophyny1quinone. UV. u1travio1et. Vis. visib1e. Xi.

(12) X11.

(13) Sec”on〃. Genera〃〃n布。duo”on. 一1一.

(14) Sec”o〃7 ＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝；. 一．1Preface. Proteins which inc1udes in enzymes are biopo1ymers which are consist of po1ymerized. L−amino acid residues by peptide bonds，and are impo血ant organic compound which are. concemed with various bio1ogica1phenomena such as metabo1ism，signa1ing，immunization， moti1ity，stmcture presewation of ce11shape，and so on．These血nctions ofprotein correspond to their stmctures．E1ucidation the p1ace，shape or chemica1property ofactive site that invo1ves. in∼nction direct1y by dete㎜ining protein stmcture1eads to revea1biochemica1reaction mechanisms eventua11y．. Revea1ing the cnzymatic reaction mec11anism is prerequisiteわr detemination and characterization ofenzymatic血nction．h the past，it has been used mutagenetic，spectroscopic and kinetics methods to e1ucidate the specified amino acid residues or regions．However，these. t㏄㎞iques are impossib1e to obsewe the movements of side chains or water mo1ecu1es．X−ray. cワsta11ography is a power制too1for dete㎜ination a1most three−dimensiona1positions of. atoms in enzyme mo1ecu1es．0bsewation sequentia1photographs such as bur−dimensiona1 movie，that is，detecting some reaction inte㎜ediates at atomic reso1ution duhng cata1ゾic. reactionandreconstitutionthcse stmctures，giveus enoughvarious infomationtounderstand reaction mechanism faci1e1y．. 一2一.

(15) 1．2Phenylet11ylami■1e Oxidase from Ar舳robacねr ψob”brm7s（AGAO）. Pheny1ethy1amine oxidase 舟。mル。加。ろααe7gZoろ伽舳ゴ∫ （AGA0）cata1yzes the oxidative deamination of2−pheny1ethy1amine（PEA），l that is the tme bioactive amine substrate，. to the corresponding pheny1acetoa1dehyde，concomitant withNH3and H202：. 十NH3＋H202. 十02＋H20 → NH2. I・I. AGAO is homodimer of∼70kDa subunits and participates in degradation amines as nitrogen source．In paれicu1ar，AGAO requires Type−2copper and2，4，5−trihydroxyphenyIa1anine quinone （TPQ）in active site．This TPQ is designated the bui1t−in co血。tor and is converted丘。m a speciic tyrosine residue posttrans1ationa11y in the presence ofCu ion and mo1ecu1ar oxygen．2’3’4 Bui1t−in quinone cofactors such as TPQ are showed in Figure I−1．. か. 。. 、。. ”. e C00H. 0. Figurel・1 Bui1t−in quinone co曲。tors． α， 2，4，5−trihydroxypheny1a1anine quinone （TPQ）；わ， cystein. tryptophy1quinone（CTQ）；c，1ysi1tyrosi1quinone（LTQ）；a，t㎜tophan tryptphy1quinone（TTQ）；e， pyn＝o1oquino1ine quinone（PQQ）．. 3一.

(16) Copper−contahing amine oxidases（CAOs）inc1uding AGAO occur ubiquitous1y in various organisms，丘。m bacteria to eukaryote，and are homodimers of∼70−95kDa subunits． CA0s，which contah TPQ in active site，Participate i血regu1ation ofpo1yamines concentration in growth，se1f−d曲nse against bacteria1in危。tion at extema1damage（p1ants），regu1ation of. intema1bioactive amines concentration（higher organisms），and so on．To date，overa11. stmctures ofho1o−CAO has a1ready been detemined丘。m舳加7た肋。o方（ECA0），5’6pea seed1ing（PSA0），7肋nsen〃αρoヶmoψ加（HPA0），8bovine serum（BSA0），9and so on（Figure I−2）．CAOs are simi1ar stmctures who1e and in active sites，however they are1ow homo1ogies between each other．ノ. 6. d. c. ／辿・. I＝igore■■2. 0vera11stmc血res ofamine oxidases（AOs）．α，ECA0（PDB ID：10AC）；わ，PSA0（1KSI）；o，HPAO （1A2V）；a，AGA0（1IU7）．. TPQ，which conc1udes in active ho1o−cA0，is generated丘。m consewed tyrosine residue，. which conc1udes in inactive apo−CAO in the presence of02and Cu．3’1o And hencc，CAOs have. two血nctions of e11zymatic reactions：oxidation of Tyr to TPQ（sing1e tumover）1L12and oxidation of amine substrates（mu1tip1c tumovers）13川5．It is necessary to revea1how two 血nctions exert br comprehend who1e eI1zymatic ro1e．. 一4一.

(17) 1．3Structura1Studies on E11zymatic Reaction ofAGAO Mutagenetic，spectroscopic，stoichiometric or structura1ana1yses ofAGAO were ca㎡ed. out to revea1TPQ biogenesis and cata1ytic reaction mechanisms．H431AH433A，H592A16and D298A17’18mutants and Ni−and−Co−substitutions．19’20Stmctures ofeach steps ofthe enzymatic reaction can be specu1ated丘。m the stmctures during the reaction which has1ow reaction rate or. the stmctures which are stopped at by using mutant，centra1meta1−substitution，i血ibitor or. substrate ana1og．However，it is meaning冊1that pursuing the stmctura1changes during the. reactiononthe‘rea1’conditionbyusingWTAGAO withPEA，thatis‘rea1’substrate． Three−d−imensiona1stmctures of ho1o−AGAO，apo−AGA021and a mmber of reaction. inte㎜ediatesweredete㎜ined，andTPQbiogenesisreactionmechanismwasrevea1edbythese stmcture changesll，in previous study By using the method of substrate soaking and. nash−coo1ing，majorreactioninte㎜ediatesweretrapped．Iftheenzymaticreactionincrysta1has much1ower reaction rate，which means minutes ord−er，than in so1ution，this tec㎞ique is so easy. and secure．Proposed cata1ytic reaction process22is shown in Figure I−3．The cata1束ic reaction. mechanism between oxidized TPQ（TPQ。、）inc1ud−ed in ho1o−AGA0and primary amine substrate to reduced TPQ（TPQ、、d）and a1dehyde product，yie1ding schi任一base which is imine fo㎜．17This cata1束ic reaction which divided into the而11owing two ha1トreactions o㏄urs by uti1izing a ping−pong bi−bi mechanism：. TPQ。、十沢CH2NH2→TPQ、。d＋RCH0（Reductive ha1f−reaction） TPQ、。d＋02＋H20→TPQ。、十NH3＋H202（0xidative ha1f−reaction）. Inthe危merreductiveha1f−reaction，theaminogroupofaminesubstrateattachesnuc1eophi1ic to the C5＝O carbony1group of TPQ。、㎡㎎and substrate schi任一base（TPQ、、b）b㎜s simu1taneous1y．Next，C，proton of substrate region is e1iminated by the protonated the C＝0. carboxy1group of Asp298which is㎞own consewed cata1ytic base and product Schi任一base （TPQP，b）的ms．Yie1ding TPQ、、d，which is ca11ed aminoresorcino1，and a1dehyde product are. subsequent to hydro1ysis of TPQp，b．On coordinating TPQ、、d with CuH，which is ca11ed. …一. E・pP・・・…f・㎜・ti・・，・・d・・t・…愉i・g… 1・・t・㎝舟・mTPQ、、。t・C・’I，. 一5一.

(18) topasemiquinone（TPQ，q）radica1fomsandCuHisreducedtoCuI，andtwostatesisequi1ib㎡um rapid1y in eac11other aerobica11y．In the1atter oxidative ha1｝reaction，CuI and TPQ，q are. oxidizedandTPQ。、regeneratesthroughiminoquinoneb㎜（TPQi．q）byo20xidation．23’24. 寸〆. 津。・・. t、∼0｛O. λ㎜・480㎜一P則. ◎. H. O. θ. Sobs打a書8Sc、耐Ibase. Product Sch耐・base. 岬㌦J. “叫釣，. λ㎜訳■425nm. λm駆出3521’■m. ｛◎ ㏄㌔. ○肥●99. Reductive. N｛. 8則＼ワ. Ha1f−reacti◎n. 。”し0 旧Y”. ㌧0. 0xidative Ha1f＿reacti◎n. ○寸 NH 0■. C凹｛1）. C川11）. ㌻、. 、岬. ｛ら 02. lmio明uio㎝e λ㎜33500m. ㎝. fOpaSem・q0・nOIle. 町へ1 λ㎜“＝436，466■1m. ＾m・nOreS0『d腕0・一TPq餉D λ㎜8310011、. Figure■．3 Results of the UV／vis absoΨtion spectra changes of during cata1班ic reaction of AGAO in each. crysta1with approximate dimension of O．4x0．3x0．1mm．The crysta1s were soaked to PEA so1ution br O，1，2，5，15，30，45，60，and120min．. Xenon is occasiona11y used as a prove for丘ndin9020r CO binding site，because of fo11owing reasons．25First，hydrophobic atom size is simi1ar to them mo1ecu1es（both ofthe van. der Waa1s diameters are about4．3，4．2and3．3A，respective1y）．Next，it is ab1e to measure anoma1ous X−ray scatte㎡ng of xenon and to detect easi1y by means of a1arge amount of. e1ectrons（about three−times）．The xenon binding sites of both ho1o−AGAO（not react）and CAOs丘。m other sources were repoれed in previous papers．25’26Two xenon binding sites were. foundinthevicinityofCuIiandTPQ。、．Fu血hemore，theoxidativeha1￡reactionhadapathway which was through not TPQ、、d but TPQ，q a丘erthe reductive ha1トreaction．24. 一6一.

(19) ㎞this work，b11owing up cata1ゾic reaction of AGAO was ca㎡ed out by using WT. AGAO and2−Pheny1ethy1amine（PEA）substrate，one ofthe most active substrate ofAGA0．1h other words，reaction mechanism with‘real’enzyme and‘rea1’substrate was revea1ed by dete㎜ination stmctures ofreaction intemediates．React nearnative condition is exceeding1y. imp0111ant to e1ucidate enzymatic∼nction．I su㏄eeded to detected major bur reaction. inte㎜ediate stmctures，which are AGAO冊A comp1ex（Michae1is comp1ex），substrate Schi旺一base，product Schi肝base and AGA0／pheny1aceta1dehyde（HY1）comp1ex by adopting time−course X−ray crysta1stmcture ana1ysis．In addition，I detemined the stmcture xenon. comp1ex（Xe−AGAO／HY1comp1ex）．. 一7.

(20) 一8一.

(21) Sec”on〃. 臥ρ馴’men制片0Ced〃eS. 一9一.

(22) Seo”on〃 II．1Purificatio■1. 11．1．10verexpressio■10fApo−AGAO I used host且。o方BL21（DE3）which dismpted two（ん〃0and肋。珂。f three cata1ase. genes（named CD03）and the p1asmid encoding the wi1d−type（WT）AGAO e㎜yme（named pEPO−02）．17’19”It is di飾。a1t to separate AGA0丘。m cata1ase，since cata1ase has extreme1y. intenseabso叩tionat400㎜whichover1ap1abso叩tionmaximumwave1en敏hat480㎜ de㎡ved丘。m TPQ．17The Recombinant apo−AGAO was ove叩roduced in cD03cεmlying. pEP0−02inCu−dep1etedandAmp−containingMg minima1medium．. 11．1．2Purificatio■10f l・lolo．AGAO. Theprec㎜sorapo−AGAOwasp舳edbya㎜oniumsu1曲te丘actionation，hydrophobic 1iquid chromatography and anion−exchange1iquid chromatography in Cu−dep1eted bu脆rs. contai㎞ng1mM DDC as the copPer che1ating SDS・PAGl≡. ”atiΨ8−pAGI≡. agent．3Apo−AGAO was converted into mature. h・1叶AGAObyb・ingdi・1yzing・g・inst50μM ○＜一 ○← CuS04的r1day．Purified ho1o−AGAO so1ution （Figure II−1），which has abso叩tion maximum at. 480㎜dehved舟。mTPQ（TPQ。、）andshowl. 「ed（Figu「eIL2）・wasconcent「atedb・Fig…11・1 u1trafi1trationto10mg／mL. Puriied ho1o−AGA0．The puri丘ed ho1o−AGAO. apPeared as sing1e protein. band. SDS−PAGE and Native−PAGE．一10一. on. both.

(23) 6. ” 0．2. 8 ε. ・2 o．1. 0. 8. 〈. 0 260. 300 360 400 460 600 650. 600 ●50 700. W8veleo￠、川m. ■＝igure■■一2. UV（7is abso叩tion spectmm of11o1o−AGAO so1ution．α，AbsoΨtion maximum wave1ength is. 480㎜de㎡ved丘。mTPQ。、；ろ，Theho1o−AGA0so1utionindicatedredco1or. 一11一.

(24) ■■．2Preparatio■10fCrystals. ll．2．1Crystallizatio■10f l・1olo−AGAO. Ho1o−AGAO was crysta11ized by the microdia1ysis method in cワsta11ization resewoir. so1utioncontaining1．05Mpotassium−sodiumtartratein25mMHEPESbu価er（pH6．8）at293 K．Crysta11ization and fo11owing substrate soaking ca㎞ed out under anaerobic conditions which. were achieved by丘11ing fi11ed．with∼99．9％N2gas in the g1ove box28and were monitored by. indicatorsofaerobicatmosphere．P1ate−bmcrysta1swithapproximatedimensionsof0．4x O．3. xO．1㎜（Fi馴re11−3）were酊。wninaboutaweek．Dia1y1i1bu廿。nlweretransfemedhto cryoprotectant so1ution which contains45％（v／v）g1ycero1br1day，because AGAO crysta1s are. extreme1y丘agi1e in case of osmotic pressure chmges，they are easi1y cracked when the composition oftheirmother so1ution is changed．. I＝igu■’e．■一3. Theho1o−AGAOcWsta1s．P1ate−bmcrysta1swereapproximatedimensionsof0．4x0．3x0．1 ㎜．. 一12一.

(25) 11．2．2Trappi■1g ReactiorI l■1termediates of Reductive ■・一a1f−reaction. Thecrysta1softrappedreactioninte㎜ediateswerepreparedbyb11owingmethod（Figure 11−4）．Bybeinglcoopedwithathinnylon1oop（φ，0．3−0．4㎜）（HamptonRe1earch），町1ta11of ho1o−AGAO were soaked into substrate so1ution contai㎡ng4mM PEA in the cryoprotect㎝t. so1ution施r5，15and120minutes at293K，anaerobica11y．Soaked crysta1s were f1ash一五eezed. in1iquidnitrogeninordertotrapreactionintemediates．. 1＾・・…舳11yミ. ．…………… P仰G・…8・・l1……一…………・・. 1官⇒国、鶯、〆1⇒. Xイay Crys屹IIog固p■1y uV〃is Spectrop110量。metry Reso■1aoce Raπ■a■1 Sp6ctroscoI＝Dy. ：. Sobs‘■．8量e ｛0＿120minD. ＝Crys屹I1iz副iOn. ：. FIash・c00Iin9＝. ESR SI＝DectrOscOpy. ，。，．．，。．．．．．．．．．．．。．．．．，．傑割．．．．．．．．．．．．．．．．．．．．．．．．．、。・. ■＝igo■’e一■．4. Preparationschemeofcrystalsofreacti㎝inte㎜ediates．Cワsta1swerepreparedingroveboxto prevent tuming over丘。m TPQ，q to TPQ。、（oxidative ha1￡reaction）・AGA0crysta1s were grown by microdia1ysis method．Fo11owing soaked cワsta1s into PEA so1ution而r0−120min， crysta1s ofreaction intemediates were f1ash−coo1ed into1iquid nitrogen．. 一13一.

(26) ll．2．3Xenon Comp1exation The crysta1of xenon comp1ex was prepared−by b11owing method．Dia1ysis button in the cryoprotectant so1ution was trεms危rred into substrate so1ution which contains4mM PEA br1. day to conve11＝to EP comp1ex（AGA0個Y1comp1ex）．The crysta1was scooped using the thin ny1on1oop and p1a㏄d in the CRY0−Xe−SITER（Mo1ecu1ar Stmcture，CXS−300）（Figure II−5），. and subsequent1y was introduced xenon gas at10MPa br45min at293K．h orderto prevent dehydration ofcrysta1飾rthe comp1exation，sma11vesse1containing mother1iquor was p1aced in. the chamber nearthe1oop mounted crysta1．The crysta1ofXe−AGAO comp1ex（Xe−HY1／AGAO comp1ex）was a1so f1ash一五eezing in1iquidnitrogen．. 一14一.

(27) I＝igu■’e．．一5. CRY0−Xe−SITER．AGA0個Y1comp1ex crysta1was introduced xenon gas at10MPa br45. min at293K. 一15一.

(28) ll．3∪V〃is Absorptio■1”easurements of Crystals. Si11gle・Crystal Microspectrop110tometry. 11．3．1. UV〃is abso叩tion spectra ofcrysta1s were measured at100K in orderto precheck which. steps of the reductive cata1ytic reaction. in. each. crysta1． The. sing1e−crysta1. microspectrophotometリ system （Figure II−6α）which comprised Cassegrain mirrors，a deuterium tungsten1amp，a CCD−amay spectrometer（0cean Optics，PC2000）and an optica1 fiber，was described in previous report．17Abso叩tion spectra ofthe crysta1s were recorded．in the. wave1en敏hrangeof250−800㎜andcomectedbyana止一b1a吐re虹ence．Fromthatwork，the maximum abso叩tions wave1engths ofthe chemica1species were signiied that TPQ。。，TPQ。。b，. TPQp，b，TPQ爬1，andTPQ．qwere480，352，425，310，and436／466㎜，respective1y・. ” 6 C榊inian @藪， Ca醐叩aomi『「o「帆、. 機軸紬、出舳！’ 一・・…叫・1ヂ＼二・・…・・…. い窟 1：1：1㌧一11111111筆・．. C榊1．・. 8. 8・、・．o. “. 〈o．2 0．1. TPg。・・’0。二．機ド. ’. じ．．．…TPQ. 4. ・・舳・・“’ 1. sqTPQ一．． ’I. ㎞ぐ’’’撃㎜、、。。. WaV61609t川0m，．・▼. uI16ar CCD｛πay s陣。trom8記r. ■＝igu■’e．一一6. UV／Vis single−crysta1microspectrophotometry．o，Scheme of microspectrophotometry；ろ，Spectra which was deconvo1uted ofchemica1species in so1ution．. 一16一.

(29) ll．4X−ray Di冊raction Experimer−ts. ll．4．1X・ray Diffraction1V1easurement and St川。ture Refineme11t. Di肝action images were recorded with X−ray syncl■rotron radiation（λ＝1．0000A）at the. beam1ines BL38B1and BL44B2at100K using an ADSC CCD detector at SPring−8（Hyogo， Japan）．hdexing，integrating and sca1ing of di附action data were canied out by using. HKL2000．29A11initia1stmctures were so1ved by mo1ecu1ar rep1acement by MOL肥R30The. searchmode1sofreactioninte㎜ediatesandxenoncomp1exstmctureswereusingho1o−AGA0 （PDB n）2CFD）and Xe−AGAO comp1ex（not react；PDB皿）1RJ0），respective1y in which a11. so1vent mo1ecu1es were removed．A11initia1mode1s were reined using㎜FMAC53L32and PHENIX，33and water mo1ecu1es were added using ARP／wA㎜．34At region around residue382 residue region，the mode1s ofTPQ。。〃EA，TPQ。。b，TPQp，b，and TPQ，q／HY1were bui1t by using the di脆rence e1ectron density maps，2F．b、＿Fc，lc，F．b、＿F、、1、，and omit map，respective1y The. mode1s were checked and rebui1t using C00T35The丘na1stmctures were va1idated using PROCHECK．36Figures were generating using PyMo1．37Cavities ofthe substrate chame1and the putative oxygenpathwaywere ca1cu1atedusingMOLE．38. 一17一.

(30) 一18一.

(31) Sec”on〃7. ∪WWsんbs0ψ”0n Sρec缶a Ofαγs崎”s dUπng Caねψ”o Reao”on. 一19一.

(32) Seo”on〃7 一．一．1Sing■e−crysta■一V■icrOspectrOscOpy. The UV／vis abso叩tion spectra changes during the cata1ytic reactions of AGAO in each. crysta1are shown in Figurc III−1．Based on the spectra of the crysta1s with deconvo1uted. absoΨtions，I危und that the wave1ength ofthe maximum abso叩tion spectmm of the crysta1. soakedinPEAbr2minwasc1oset0352㎜，consistentwiththatofTPQ、、b．Twopeaks，436 and466㎜，arosewiththepassageoftime；peaksat60min，simi1art0120min，were436and 466㎜，whichwereconsistentwiththoseofTPQ，q．Theseresu1ts suggeltedthatAGA0in crysta1s reacted with PEA during the timc course舟。m2to60min．In previous studies，the rcactive time in so1ution was60ms，whereas this time in crysta1s was60min．Therefore，the cata1束ic reaction time ofAGAO in crysta1s was pro1onged to as much as∼6．0×104times the reaction rate in so1ution duc to the in且uence ofthe protein crysta1ie1d．. 一20一.

(33) 120 60. 45. 。。∼. 1・ぷ. 00 6 0 ．o − Oω. ・ダ 2. ○ く。）. 1. ε 里。. “. 0 300. 400. 500. 600. 700. Waveleogt川nmD ■＝igure Il．■1. Resu1ts of the UV／vis abso印tion spectra changes of du㎡ng catalytic reaction of AGAO in each. crysta1with approximate dimension of0．4x0．3×0．1mm．The crysta1s were soaked to PEA so1ution br0，1，2，5，15，30，45，60，and120min．. 一21一.

(34) 22一.

(35) Sec”on〃γ. S炉Ucω胴〃Changes ofRedUc”γe〃a仕Reac”on. 一23一.

(36) Seo”on〃γ 1V．1Structure Determinatio11s Isucceedtodete㎜inethc丘vestmctureswhichweretrappe舳rthereactiontimewas5， 15and120minutes at atomic reso1ution．These crysta1s were named RT5，RT15and RT120， respective1y．Fuれhe㎜ore，thesc crysta1stmctures correspond to the Michaehs comp1ex，the substrate Schifトbase，the prod−uct Schi任一base and the AGA0個Y1comp1ex，respective1y．The statistics of stmcture reinements are a1so given in Tab1e lV−1．Each stmctures were reined at. around2A reso1ution in space group C2with unit ce11dimensions aboutα＝192，わ＝62，c＝ 158A，β一117◎（‘1・・g・C2・・11’2’）．. 一24.

(37) Tab1e lV−1. S肋ti前icsofωacoll㏄tion㎜d岬sωlo卿hicrd㎞㎝㎝tofthe岬sta1so水d㎞PEA㎞rO，5，15，120mh （＾）R’I’0. ｛8）RT5. （C，O）RT15. （E）RT120. D8t8co11odio■1 Un“COII dim008iOn8 a（A1. 192．75. 191．57. 190．97. 191．21. 川A）. 62．79. 63．56. 63．64. 63．42. 剛A，. 158．01. 157．67. 157．56. 157．61. b｛dog）. 117．56. 117．10. ”6．82. 117．05. Spac6group. C2. C2. ’■’0t8I I’1・、・mb670f Ob80【’di008. 州99227. 仙3647. c2 631254. C2 531283. fo−8111umbo■一〇niqoo l’♂bctio■、8. ‘170079｛23033）. 83982（7733）. 93540（9425）. ‘132474（18972）. R0801u−i㎝㈹. 58．93・’1．74｛‘1．83・‘I．74）. 50．00・2．20（2．28−2．20）. 50．00・2．10（2．18＿2．10）. 61．43・1■90（2．00＿1．90）. Complot011098（％⊃. 98．9（92．6）. 97．4（90．3）. 94．8（96．1）. 99．7〔98．7）. Rm．。㈹. 6．’■（32．8）. 6．5｛27．6）. 7．0（36．3）. 7．3｛35．9）. ”o岬1ic吋. 7，1（4．6，. 5．3（4．6）. 6．8（5．9）. 4．0（3．8）. ”oω. 8．‘■｛2．3）. 23−6（3．9）. 32．8（6．5）. 7．3（2．0）. R0801“i㎝㈹. 50．00・1．74. 50．00・2．20. 50．00＿2．10. 50，001‘1．90. R…資㈹. 16，8. 16，6. 18，8. 17，1. R㎞㈹. 19，9. 21，5. 24，8. 21，0. ∼o固g●8地do剛A）. 21．9. 32．3. 28．9. 27．4. R前n㎝旧0bddbtiC8. rm8d07ia−i㎝fromi’081㎜lo08. 8㎝d1oog仙8㈹ 8ood809168（dbg）. 0．012. 1．4. 0．019. 1．8. 0．021. 2．0. 0．015. 1．5. †Rm甘g。＝Σ＾Σ、14、、一〈4〉1／Σ后Σみ、・ ‡Rw誠＝ΣllF．1−lF，ll／ΣlF．1．. §R吐。＝孔趾fora叩mx㎞ate1y5％oftlle rei㏄honsth就were exc1山d．ed．丘。mthere丘皿em㎝t．. いべ.

(38) 1V．1．11・1olo−AGAO Structure. Idetc㎜inedRT0（notreact）tocompareotherreactionintemediates．RTOwasreinedat 1．74A reso1ution in space group C2withunit ce11d．imensions o：192．75，ろ＝62．79，c＝158．01 A，β＝117．56o which was be1ong to‘1arge C2ce11’21． The active site stmcture ofRTO is shown in Figures lV−1o andろ．The entrance and bottom. ofthesubstratechame1hadnegativecharge．And，themidd1eofthechame1washydrophobic． IntheF．b、＿F、、1，omitmap，twopositivepeaksof3．5o1eve1wθreobservedinactivesite．Fo㎜ ofthe e1ectron densities in the vicinity ofcopPer ion c1ear1y showed the TPQ。、（occupancy1．oo）． TPQ。、does not coordinate with cupPer ion，name1y，it obtained cata1ytica11y active“o紙一。opPer’’. conbmation．Tyr296（ca11ed‘gate’residue）1ocatedbo廿。mofthechame1，openedthechame1 缶。m extema1so1vent to TPQ。、and Phe105and Leu358’（named‘cap’residues）opened the. entrance ofchame1．AroundCuion，equatoria1water（We），His431，His433，His592andaxia1 water（Wa）coordinated with Cu ion，whichhad a distorted square pyramida1geometW（Figure IV−1o）．. 26一.

(39) トベ. ｝. ” fPQ。貨. u3581. Asp298. 。。。…I. Cu τyr296. 33. fyr284 1・．□S431. 用s592.

(40) c. TPQ㎝382. Wa. ・一・is433. ｛＾Xial，. We ．．・’’. @. ・ 41≡qOatOria■，. ”is43. His592. ■＝igure lV−1. Structures ofRTO ofAGA0．The Cu atoms and watermolecu1es are shown co1ored brown and red， respectivelyα，The F．b、一F、、1，map contoured at3．5o in active site；ろ，The su曲。e around the. substratechame1；c，Stmcturearo㎜dCui㎝，equato㎡a1water（We），His431，His433，His592and. axia1water（Wa）coordinatedwithCui㎝，whichhadadistortedsquarepyramida1geometry．. 一28一.

(41) 1V．1．21Vlichae1is Comp1ex ofAGAO and PEA A㏄ording to the sing1e−crysta1microspectrometワdata，spectmm ofRT5was simi1ar to. thatofRT0（notreact）．Thisresu1tindicatesthatTPQ。、hasbeensti11b㎜edandthecata1束ic reaction does not staれ．The cワsta1stmcture ofRT5is shown in Figure IV−2o．In the F．b、一F、、1，. omitmap，榊。 positive peaks of2．6o1eve1wereobsemd inactive site．Fo㎜ofthe e1ectron density in the vicinity of copPer ion c1ear1y showed the TPQ。、（occupancy1．00），and another. 1arge e1ectron density in substrate cha㎜e1was suggested PEA（o㏄upancy0．70）．TPQ。、does not coordinate with cupper ion（“o肝一。opper”c㎝fo㎜ation）same as RT0，PEA1ocated in the. substrate channe1（Figure1V−2わ）and fo㎜ed hydrogen bond to Asp298with2．6A．The aromatic ring of PEA was丘xed in hydrophobic pocket which was configured by side chain. （Figure IV−2o）such as Phe105，A1a135，Pr0136，Leu137，丁叩168，TW296，Tyr302，I1e379，. G1y380，Asn381，Phe407，Leu358，andTΨ359，．Tyr2960penedthe chame1缶。m extema1 so1vent to TPQ。、same as RT0．However，Phe105a血d Leu358’moved as cap ofthe chame1 Pa「tia11y，in cOntrast to RT0．. 一29一.

(42) か. ” TP◎舳. 、ヌ㌔. PEA ’・。. ・ペ. ！．. ＾sp298. 1誰．げへ・ 1 、戸、. 文. マ粥. Cu. Tyr296. 1・1is433. τyr284. ㌧. Substrate C■1am1e1. 1・■is431. 1・・iS592. ◎. n.

(43) c ’†. ヂ、〃. ㍑. τ巾お9・. 桑一. ＼い了. ノ、. 、＼）. 、. 葉. 1−8035. 燃. ⑫168. 、／. ∼ ．，㌧←. ！と. 。・ …1土・. ふ. O1y380. ●. ㌣凱醐. 7（み．、、. ㍗㌻. Co. ＼・. ＼．. （合. ■＝igur61V・2 Structures ofRT5ofAGA0．α，The F．b、一F、。1，map contoured at2．6o in active site；わ，The su誠ace. aromdthesubstratecba㎜el；o，Stereo㎡ewsho㎞㎎hydrophobicp㏄ketinthesubstratechamel．. 一31一.

(44) ■V．1．3Substrate and Product Sch耐一bases. hthepreviousrepo血，IbundtheRT15stmctureat2．1Areso1utionわ㎜edtheproduct schi任一base in subunit B ofAGA0．28chemica1stmctures ofTPQ、、b and TPQP，b17were shown in. Figure lV−3．This was the firstrepoれ。fWT AGAO andPEAsubstrate．Inthis paper，Ircpo血ed. thestmctureofsubunitAofRT15andthedi脆rencereactionrateofsubunitAandsubunitB． The crysta1stmctures ofRT15ofeach subunit are shown in Figures IV−4and IV−5．According to 趾ting mode1s with the F．b、＿F、、1，omit map contoured at2．5o I危und substrate and prod−uct. Schi任一bases which were b㎜ed in subunit A and subunit B，respective1y．O㏄upancies of TPQ、、b and TPQP，b were1．oo and．1．oo，respective1y・Stmctures ofTPQs．b and TPQP，b with bond. ang1eswereshowninFigu．reIV−6．Bondang1esofNandC，was127．0．and114．7ointhe substrateSchi任一base．hcontrast，bondang1esofNandC．was113．1oand125．3ointheprod．uct Schi任一base．The C，position of the substrate Schi任一base was c1oscr than that of the product Schi旺一base，and−the d．istance betwecn C．ofthe substrate and product Schi任一bases and Asp298. were2．71Aand3．91A，respective1y．Inthebothsubunits，thesubstratechame1containingthe Schi旺一bases had neutra1charge．The‘gate’residue opened the chame1same as that of the. Michae1is comp1ex，and the‘cap’residues closed the entrance of the substrate chame1 comp1ete1y．. ”. 6. λ. ㌔. o、共。べH0二票。∼cρ 。．．．．．．山H・ θ. 。＼・. ○. I＝igure lV・3. Struc血ra1b㎜u1aeofSchi住basesconsistingofTPQandPEA．α，substrateschi胆basestmcture；わ，Product Schifトbase structure．. 一32一.

(45) か. ”. いい. TPO。。b ん. 艦. ㍊. 、＝，満1一’. 着. ＾Sp298. 、「。㍉ L 寺. 、．，. 略融1. Cu Tyr296. 1・1is433. Tyr284. ㌧. His431 1・・iS592. I＝igu胞■V・4 Stmctu爬sofchai皿A inRT15ofAGAO。α，TheF。㎞一尺心map contoured．前2．5o・i皿active site，わ， The sur曲。e around the substrate chamne1．.

(46) か. ” b. 瓢望. Co Tyr296. 33. ＼＞. Tyr284 1・■1s431. 1・1is592. Figu爬■V−5 Stmc血爬sofchahBi皿RT15ofAGA0．α，TheF；b、一F、＾。map◎ontoured前2．5o・inac廿ve site，ろ， The sur曲。e around the substrate chaIme1．. 寸㈹.

(47) c. ”. いい. 127．0 114．. 6. （. ”. ク. I＝igure■V−6. S化・・舳…「”q・・md1PQ。…fAGAO・α・St㈹・i・w・m…㎞・dm・d・1・「TPQ坤・h・㎜wi叫・・一上；小㎜p・・皿t・皿舳t2・5σ・か・s㎜1e as hαbut viewed・血。m the di工ection㎞para11e1with血e TPQ ri皿g，o，ste肥。v1ew of血e爬O皿d mod・e1ofTPQ帥show11w皿1 F；h、一尺。■、㎜p conto山ed．at2．5o’，a，Same ashc1）ut viewed耐。mthe血ectioni皿para11e1withthe TPQ｛g．.

(48) 1V．1．4AGAα1−lY1Complex（Complex of Reduced AGAO andProduct）. A㏄ording to the sing1e−crysta1microspectrometry，the spectmm of RT120was. congruencewiththatofTPQ．q，thatis，itwassuggestedthatsemiquinoneradica1was施medin RT120．ThestmctureofRT120dete㎜inedatl．90Areso1utionisshowninFigurelV−7．InF．b、＿F、、1，omit map，two peaks of3．0o1eve1are obsemed in active site simi1ar1y to RT5．It was. dete㎜ined an e1ectron density1ocated TPQ。。Position was dehved丘。m TPQ，q（occupancy 1．00）and another one was derived丘。m HY1（o㏄upancy1．00）．I conc1uded that RT120was. 丘na1stateundertheanaerobicenviro㎜entandthereductiveha1f−reactionofcata1束icreaction tc㎜inated comp1ete1y in RT120・TPQ，q coordinated to copPer ion with the coordination. distanceof3．OA，name1y，it的㎜edcata1束icamyinactive‘‘on−copPer”con的㎜ation．HY1was. 1ocatedatthe samepositionwherePEAexistedinthe substrate chame1inthe RT5．The‘gate’ residue c1osedthe chame1，in contrast to㎜5and RT15，which was synchronous with nip of TPQsq，and the‘cap’residues remained to c1osethe channe1．. 一36.

(49) 6. ”. トい. TP◎。。. HY1. 。．●. 洲. ＾sp298. Co Tyr296. ●. ・．. 、. 一・lis433. τyr284. His431. 1・■1S592. ■＝igo記1V−7. S血Tctu記s ofRT1200fAGAO．α，The凡b、一九一。㎜p contour記at3，0σi皿ac廿ve si屹，ム，The su曲。e am㎜d− the s1u［bstrate channe1．.

(50) 38一.

(51) Sec”onγ. D7oxγ9en−b肺ψng sκes bγ∪s7ng Xb Co㎜ρ〃ex. 一39一.

(52) Sec”oπγ V．1Xe−AGAO〃・lY1. Complex. Ia1sosu㏄eededtodete㎜inethestmctureofxe−RT120at1．90Areso1ution（Figuresv−1 and V−2）．The stmcture was cong㎜ous with that of RT120except xenon−bindi皿g sits and the. conbmationofTPQ，q．Thestatisticsofstructurereinementarea1sogiveninTab1ev−1・. Table V−1 Statistics ofda吻。oll㏄tiom2md㎝ys制1og螂p血。 rd耐6memt ofXe−RT120. （■＝）Xo＿RT120. 0剛8cOlI●di0・1. U0，CO11“m○・18i008 8（A）. 191．17. 剛AD. 63．98. o（A）. 157．60. 6（d剛. 州7．00. S岬㏄groΨ ．rOt8I■’10m6●r Of Ob80■’、’｛0n8. C2 790105. ．■■0t8100mb●r ul’Iiq0●【6610C−iOn8. ‘122827（1‘1989）. R●901“㎝（∼. 50．00＿1．90｛1．97＿‘1．90）. Comp1市m88㈹. 90．5（89．0）. ∼。㈹. 7−0（42．5）. m岬1ici∼. 3．3｛3．’1エ. ”o（o. 35．1（5．4〕. R0資00m6■1お8tdi8tiC8 R080■・、■tio■1（AD. 50．00＿‘1．90. R舳（％）. 17，4. R㎞（％）. 21，5. ＾）6㎜g●84ador8｛A）. 26．0. ・・m8do）i8tioo fl・om i・＝・081”8■u08. 8㎝d16㎎仙8（AD 8ood8・、9169（dog）. 0．015 1．5. †R。、、。．一ΣゐΣルー／4／1／ΣんΣみi・‡R。批＝ΣllF．1−1尺11／ΣlF．1. §R∼。＝Rw。｛的r棚mx㎞．晩1y5％of血e rei㏄吐。nst1耐wereexc1uded丘。m血ere固皿em㎝t・. 一40一.

(53) Xenon atoms were bund four bind−ing sites per a subunit and these positions were in. accordance with bur of seven sites of the previous Xe−AGAO comp1ex（PDB皿）1RJO）．25 （Figure V−1）The crysta1stmcture ofXe−RT120is shown in Figures V−2o andろ．The F．b、＿F、、1c. omit map contoured at7．0o．One ofthe sites（Xe6（o㏄upancy0．30））was危und in the vicinity ofCu ion and the“inland lake”fmed with solvent waters，and was1ocated hydrophobic region， that is“anteroom”，26’39which was constituted by seven amino acid residues，Tyr384，A1a400，. A1a402，His433，Phe435，Leu590and va1607（Figure v−2o）．The amino acid residue ofTPQ 382had two con危㎜ations which were TPQ，q and TPQi．q which比㎜ed“on−copPer”and “o肝一。opper”con此㎜ations，respectively（o㏄upancies O．69and O．31，respective1y）．The distances between xe6and Cu or TPQ，q were7．6A or8．5A，respective1y㎞add−ition，both the. ‘gate’residue and the‘cap’residues remained to c1ose the channel comp1eteIy，and HY1 remained1ocated inthe substrate chame1（o㏄upancy1．00）．. 一41一.

(54) か. ”. Xe4 Xe6 ：．Xe1： 3 ： … 8 ：Xe5. Xe8 6 Xe5 ■ ● 、● ・ 2 8 Xe7 、一・・ザ●・. ・ ○○ ・・. 子．㌧∼○ ・．一一㌧一一● ，．ノ●．. ∼一ポ∴ゾ∴㌧、. あ. ・. ’’↑∴三∼ ……. 。、3. ：…. ぺ4宮ぐ∵けピ・純ン、. ‘. 茸｛∴1（’ ，4C・㌧…. べ∵夢為∴吋∴ヂ、㌧：. ’ち. Xe6. ：. ：・㌧. 3. ： Xe3. ・. Xe2. ’. Xe7. I＝igu爬V−1. X㎝on−bhdingsitcs ofXc−RT120andXc−AGAO．σ，X㎝㎝北h曲㎎sitcswcrc sho“mascyansph㎝帆Foursi眈sw㎝池㎜dpcr sub㎜1it om Xe−RT120，that is Xe−HY1！〈G〈O c㎝nplex，わ，Xen011−binding sites w6re showl as go1d sphe1r齪一Sevem s他s w町e. 勉md−per㎜b㎜itonXe−AGAO，th砒isXe−ho1o−AGAO comp1ex（PDB lD：1RJO）．. べ寸.

(55) い寸. か. ”. HY1. 込. TPQ。。. ＾sp298. ．C. ﾋ炸6. Tyr296. 一一1iS433. Tyr284 1…S431. 1一一is592. ㌧.

(56) c ・・lY1. ○叫. 、、、舳. fPQ382 ／. 一が一. ／！P・1e435. @. レ. ・．、、研、. ’、5．0. 5・1＼、 4少 His43ガイ．3：9・＾1・400. 、崎、、、。。凹、鰯簑以・. ．．．．1鴫、. a一. ’レ／. ’. ・■eu590. H1s592. 1＝igure V・2 Strlユ。tures ofXe−RT1200fAGA0．o，The F．b。一F。。1．map contoured at7．0σin active site；わ，The su曲。e aro㎜d the substrate chame1；o，Stereoview ofthe xenon−binding site near the active site surrounded hydrophobic side chains．. 一44一.

(57) 一45一.

(58) 一46一.

(59) Sec”on V7. D7souss7ons. 一47一.

(60) Sec”on V7 V一．1Reductive■一一a1f・reaction. Vl．1．1Reaction”echanism ofWTAGAOwith PEA Stmctura1changes in active site during the reductive ha1f−reaction using WT AGAO and. PEAwhichistmesubstrateofAGAOareshowninFigureVI−1．Isu㏄eededtodete㎜ine的ur m勾。r reaction inte㎜ediates，Michae1is comp1ex（RT5），substrate Schi任一base（RT15−A），. productSchi任一base（RT15−B）andAGA0／HY1comp1ex（RT120）．Ia1sosu㏄eededtodete㎜ine stmctures，contro1（RT0）and−Xe−AGA0／HY1comp1ex（Xe−RT120）． 0n the basis ofthe resu1ts in this and previous works，17’28the reductive ha1f−reaction with. WT AGAO and PEA is proposed as b11owing mechanism．At irst step of the reductive ha1トreaction，PEA which is protonated at pH6，840is pu11ed into the negative1y charged. substrate chame1（stepsα→わ）．Inthe RT5，Ihave detectedthe Michae1is comp1ex usingWT AGAO with PEAforthe丘rst time（stepわ）．PEA1ocates in substrate chame1ofeach subunit with high o㏄upancy．As的r PEA，the aromatic ring fixed by hydrophobic pocket in the vicinity of active site and amino group bound−to the side chain of Asp298．It is considered that the. mc1eophi1ic attack ofamino group to c5atom ofTPQ ring is o㏄urred easi1yby immobi1ization. PEA（stepsろ→o）．In consequence，substrate Schi肥base is b㎜ed between TPQ and PEA （step o）．H，of TPQ、、b binds to Asp298in order to be deprotonated by Asp298，and the. confomationoftheTPQ。。bchangestoTPQP，bbyreductionofTPQhng，subsequent1y（stepsc →の・HY1is re1eased by hydro1ysis ofTPQp，b with water，and reduced TPQ coordinates with CuII in order to reduce to CuI（steps a→e）．Therea丘er，mo1ecu1ar oxygen is provided in the. vicinityofCuI，andCuIisoxidizedtoCuIIbyimer−spheree1ectrontransおrmechanism（stepse →力（Section VI．3）．. 一48一.

(61) mi. ”. P買1鮎σD、、、三磁、葱tm州、T、、二繕. RT0・. ■■1，EA1 ’. P1…＾. キ. ＾Sg. ．. 2・・、・，tへ∼. ．メ. †1｝一ぼ. 二蛤“. ヂ. ヂ. ㌧0. Hi64 ■is431. 宇、ミ桑・. 麟τ∼. く．2. fW296. fW284. 49mitmap2・5σD. 92. Michae1is Comp1ex. Substrate Schiπ・base ↓. 岨舳岬㍗崎、勢．満州 ●. 占. ←. ’ ｛一随’。孤、 ‘. 06. Xe−AGAO用Y1Comp1ex. d RT15．8. 0. ．9叩耐map2・5σ，. 蟻・蟻TPQゆ. 、）・3. …一㌫ノ，. 淳炉一’炉. ■ 、3，0. 、ソダ. AGAO川Y1Comp1ex. ヂ ProductSchiff−base. I＝ig叩e V．一1. Aけive site X−my snapshots during血e爬dudive h虹frea㎞onusing WT AGA0町1d PEA，t1㎜t islme s皿b血ata1］二1e s1コmctures ofoontro1. （RT0），MichaeHs comp1ex（RT5），sub血娩Sc㎜トbase（RT15−A），prωuct Schi岱一b鵬（RT15−B），AGAO！HY1◎omp1ex⑭T120）㎜d Xe−AGA0／HY1oomp1ex（Xe−RT120）were show皿鎚α，う，c，♂，“nd．ス説p㏄血ve1y一. α 寸.

(62) In previous work，stmctures ofTPQ、、b and TPQP．b were detemined in D298Amutant. （FigureVI−2α）．17’41D298Amutanthas1owactivityby∼1060rderascomparedwithWTAGA0， because t11is mutan1t1acks ofAsp298，that is the consewcd cata1束ic base．In addition，Tyr296. movedbacksideofTPQ。、whichwasthepositionofAsp298．Therebre，conbmationofthe substrate ch孤me1was changed and stmctures of TPQ、、b and TPQP，b were a1so changed as. comparedwithWTAGA0（Fi馴resVI−25ando）．Mhemore，Leu358，which1ocatedatthe capofthesubstratechame1openedinthestepofthesubstrateSchi旺baseofD298Aincontrast to the same step ofWT AGA0．Thus，it was hdicatedthat more precise stmc血res ofreaction. intemediatesweredete㎜inedbyusingWTenzyme．. ”. 、，. fPQ㎝382 グ. 、. ● ト. Asp298’ Ala298. 一50一.

(63) 6. 。. 津淵. P−1e105. 〉 f叫。6. f閂舳. Co fy・296. ＾■3298’ ＾sp298. Figure Vl・2 Stmc血ral di脆ren㏄s in active site ofWT AGAO and D298Amutant．o，Stereoview ofthe active. site structure ofWT AGA0（green）and D298A（ye11ow）．The Asp298positi㎝was rep1a㏄d by Tyr296in D298A（red circ1e）；わ，SupeΨositi㎝ofmode1s ofTPQ、、b in active site ofwT AGA0 （red）and D298A（ye11ow）；o，Supe叩。sition of mode1s of TPQp，b in active site of WT AGA0 （magenta）and D298A（orange）。. 一51一.

(64) The coordination geometries of Cu are shown in Figure VI−3．In RTO，equatoria1water. （We），His431，His433，His592andaxia1water（Wa）coordinated with Cu ion，which had the distorted square pWamida1geometワ，and coordination distances were2．0，2．0，2．1，2．0and2．7 A，respective1y．In RT120，in contrast，Cu ionhadthe tetrahe血a1geometry，and the coordination. distances ofHis431，His433，His592md TPQ，q（axia1position）were2．2，2．1，2．1and3．O A， respective1y．h active site，ho1o−AGAO has a Type−2copper which easy changes舟。m diva1ent. tomonova1entinthis1igandingenviro㎜ent，andva1enceofCuisab1etoestimatebydistances to1igands and coordination geomet㎡es．The coordinating distance between Cu and axia1 position ofRT120was0．3A1onger than others which suggested that Cu ion is h monova1ent． In conc1usion，it is considered that TPQ肥d Produced by hydro1ysis of TPQP，b f1ips and coordinates with cun，and TPQ，q and CuI are produced by the intramo1ecu1ar one−e1ectron trans危r丘。mTPQ、、d to CuII．. e. ” H2◎lAxial）. TP◎、。岬・l1 9. 9. 13．0. ：2・7H。◎. 。i、。舳三11．・素。1・…t・・i・l1 、．；P炉、i、、9、. 1⇒. ■・Iis4330三’1：. 、；ρ舳i、。。。 ○. ○. ■一1is431. 1−1is431. TPO・g｛30） Wa−2．7，. Hi3433. W●. ｛2．”＼／12・01 C0. 12．01. −2．”＼ Cl』. ／＼. ！＼一’lis431. 1・．i8433. ・一・is592. 12−01. ．■1・9431. 12．21. ・一1・s592. 12・”. ■＝igure Vl・3. The coordination geometries of Cu．αhad the distorted square pyramida1geometry of RTO and e. had the tetrahedra1geome町。f RT120．Counts in parentheses indicate coordination distances in 1ower scheme．. 一52一.

(65) V1．2Di冊erence of Reaction Rate between Eac11Subu11it h RT15stmcture，it was detected the di脆rent stage as TPQ、、b and TPQp，b in each. subunits．Itwasa1sofoundthatthestageinsubunitBadvancedthanthestepinsubunitAof each crysta1（data not shown）．. Vl．2．1Structural C11ange ofArm1 ThesubunitofAGAOhastwoβ一ribbonams，thatisA㎜I（residues348−369）andA㎜II （residues453−474），21andbothams1ieinanothersubunits（FigureVI−4）．Thesea㎜sprobab1y. paれicipateindimer危㎜ationbutessentia1ro1eoftheamsisunceれain．Ithasbeensuggested thatノ㎞n I paれicipates in access of substrate to the active site because it is1ocated near the. substratecha㎜e1．21A㏄ording1y，Ifocusconb㎜ationa1changestheA㎜Idudngthereductive ha1f−reaction（Figure VI−5）．The RMSD va1ues of Am I among RTO and−others are0．11O−. 0，168A（average0，130A）．㎞contrast，the va1ues between RT5，RT15or RT120and each others were0，079−0．113A（average O．099A）．Leu358，is the biggest movement residue in the. Am1I，the distance of C，atom of Leu358，among RTO and RT5，RT15or RT120is apProximate1y1．0A．It is exp1ained that Leu358，is1ocated the entrance of substrate chame1 and f1ipped arising hydrophobic interactions with aromatic ring of PEA or HY128during the reductive ha1f＿reaction．. 一53.

(66) 。．Arm1｛348，＿369’）（、○ ．●. 一. Sobst固te Cha■1■1e1. ○. ○ ／. u358，. ．．. Arm11 （453，＿474’）糺．・. べ. ゴ. 勤. 。. 衝河. 汐. ぐ一一一導．ク！. 〃Q3幸一 ’ノ ’. Co. 蝋一・一㌔. ■＝igore Vl・4. Thestmctureinth州。inityofthesubs忙atecha㎜e1．A㎜Iisdra㎜orangecaioonmode1andLeu 358’is1ocatedatthetipoftheA㎜I．Thesubstratecha㎜e1whichisdrawnmagentasu曲。e．. 一54一. 華1.

(67) Arm1｛residues348’＿369，D. ・…. PE＾川一1Y1. i. 奥. Leu358’ Substrate CI1aI1ne■. P11e105. ■＝igure Vl・5. The s肺。血ra1change ofthe A㎜I dudng the reductive ha1トreaction．Leu358，nipped arising hydrophobic interactions with aromatic ring ofPEAorHY1during the reductive ha1f−reaction．. 一55一.

(68) Vl．2．2Structural Di冊ere11ce in Active Site of RT0. WhenI施。usedonstmctureofRT0，Ibundthedi脆renceofstmcturesoftheaminoacid residues in substrate chame1．A㏄ording to supe叩。sition the stmctures in active site ofboth. subunits，main chains ofboth subunits are essentia11y identica1．The RMSD va1ue of C，atoms. betweenchainAandchainBiso．352A．However，Phe105，Asp296，Asp298andTPQ382are changed，whichis showninFigureVI−6o．Shapesofbothsubstratechame1s aredi脆rent丘。m eachother．Thecap ofthe substratechame1isc1osedpa廿ia11y（FiguresVI−6ろando）．And，the substrate chame1reaches the vicinity of Asp298trough the backside of TPQ in chain B．. However，the chame1in chainAis deadend atTyr296and above ofTPQ，anddidnotreach Asp298（Figures VI−6a and e）．Name1y，it is suggested that the residues in the active site. b㎜cdsuitab1econb㎜ation的ra㏄essingandixationofPEAbe的rcaprotonistrans胎med 丘。m PEAtoAsp298in chain B．. ”. s蝋. d｛u鮒、熟 Asp298 Tyr296. 56一. TPQ382. ξ.

(69) か. c トい. ．敷？、ド・帥 ’. 鼠 ○● ○ ● ｝○ ｝ “一. 1ノ. 蜆. I. −. r. 閉・105評. ■ 1 ■. 鵜γ. ダ. ・。. ○ ■. 。慶’．三. ・島. ＼. 一○. む ○ ■ 、. ●一. 瀬つ1｛、1蓬二葦. 騨：. d。、、、、、1、，．．. 機「・. 7. e Substrate C■1a■1rleI. ㌔、. ㌔. 斤 L即358’. …句ヨ. Arm1. ＝携pQ382 ，・. T、、296》払。．！0C， Asp298. ！r. ●・・. ＼. ＼. ■＝igure Vl・6. Stm伽ml di価げ㎝c閑b勧ween each subuni也．4Sup岬。sitionthestmc化u欄㎞active site ofboth su．bunits．The color ofstickmode1s ofch心A㎜d cha㎞Bπe. gr町mdg㈹n，r岬㏄tive1y；か，The stmcture ofthe㎝㎞m㏄ofthεsub討mt6chamel h chain A；c，皿舳m伽爬。舳e㎝㎞m㏄of出e㎜b冒㎞t6ch㎜el in chainB；a，The舳伽reatthebo㏄omofthesubs㎞techa㎜elinchainA，e，Thestm伽r“tthebo砒。mofthes皿bstmtech㎜nel inchオnB．.

(70) Based on these resu1ts，it is suggested that the reductive ha1f−reaction staれs when PEA. entersandis丘xedinthesubstratechame1whentheactivesitestmctureinchainAadoptssame con肪mation in chain B bybreathing motion．Therebre，I conc1uded that the di脆rences of reaction rate between each subunit by this time1ag．Moreover，because the cata1皿ic reaction time ofAGAO in cIysta1s was extended to∼6．0×104b1ds the reaction rate in so1ution owing to the e脆。t ofthe protein cWsta1丘e1d，28I succeeded to detect the both Schif−bases in same R15 crysta1．. 58一.

(71) V1−2．3−V1o1ecular Packing of RTO i■1Crysta1. In the previous section，the di価erence ofreaction rate between each subunit is described． It is proposed that this consequence is attributed to mo1ecu1ar packing or the ha1f−of−the−sites. reactivity．42’43’44h this section，the crysta1packing was eva1uated by using RTO cワsta1，and the. other crysta1s are a1most isomo印hous to the RTO crysta1．The packing of RTO with six. symmethca1mo1ecu1es，A1，A2，A3，B1，B2and B3，are showed in Figure VI−7．A subunit of RTO interacts with three mo1ecu1es，suc止pattems ofinteraction are same as危㎜s II and1V crysta1s in the previous report．45A1／B1interacted−to surface ofthe D2domain（residues9−91）. and A3／B3interacted to surface ofthe D3domain（residues103−203）．A2個2interacted to1ower. paれ。fthe D2，D3and D4domains（residues229−623）．A1／A2／A3interacted．to chain AofRT0 （centra1dimer）and B1／B2／B3interacted to chain B．Any mo1ecu1es interacted with the same. chains ofRT0． The symmetrica1mo1ecu1ar interfaces did not1ocate at the vicinity ofthe entrance ofthe. substrate channe1and the stru−ctures ofthe interface were di脆rent舟。m each subunit（Figure VI−8）．It was revea1ed that the stmcture di脆rences which arose舟。m di舶erent networks of hydrogen bonds，sa1t bhdges and hydrophobic interactions丘。m inter曲。es to the active site. existed between subunits．Hence，I conc1ude that AGAO has di脆rent reaction rate in each subunit in the crysta1by reason ofthe di脆rent subunit stmctures which arose缶。m variation of. symmetrica1mo1ecu1ar packing．It is remaining that there is some possibi1ity of symmetrica1 mo1ecu1ar packing consisting of the recognition of stru−cture di脆rence which arises to the h・1ト・トth・一・it・・・…ti・itγ42・43・44. 一59一.

(72) ”. CI1ai−18. 協鵡滞瓢. 、、滋洩家. 霧. 電箏. 〃. 1耳 CI13in8. 「．∵j∴．、＾・l. 1奴々1沽. ■ ’. ∴㌧か. 。二，∵婚、。二. 、、＝饗令・・. @ボ峠㌦. 薦二二，、. 冬。〆 ■ ．㌧■呂叶. ，パく一時㌧・. ㌻碍. か. 1耳. ＾3. I＝igure Vl・7. Packing ofRTO with six symmetrical molecu1es，A1，A2，A3，B1，B2and B3．o，The pattem ofthe packing with symmethca1mo1ecu1es；ろ，The inte曲。e wi血symme㎞ca1mo1ecu1es．. 一60一.

(73) ” Sobst胞te C■1anIle1. 冶骨ポ’ノブ. ＾1’8. ＾3’83. ＾2’82. ・、、. ＾ctive Site. 1ギ. ＾2’82. 6 Substrate Chan■1el @ ノ＾・. ．・. ’’. と. 勤. ε. 〃、〆・. ＾ctive Site. 。 ’’. ﾞ．加・・．．．03. 山．幸．. A’. ：. 珊、だて。。，. 1三2. と・．し．・・．. ．・. ･. c. ■＝igor6V1・8 Stmctural di脆rences ofinter胞。e．o，The interf乞。es ofc11ain A，chain B and both chain co1or with. ye11o“orange and red・respective1y；ろ・o・SupeΨoses ofchainA（ye11ow）and chain B（orange工61．.

(74) V1．31nitia1Step of Oxidative1・la1f・reaction. V1．3．1Pat11way of Provided Molecu1ar Oxygen. The oxidative ha1f−reaction b11owing by the reductive ha1f−reaction is the step of TPQ，q. reoxidation．24Xe−RT120was not comp1ex stmcture ofxenon and RT120but mixturc stmcture ofTPQsq and TPQimq or TPQ。。・That is，it was trapPed initia1step ofoxidative ha1f−reaction・This. is caused by transpoれation scooped crysta1s丘。m g1ove box to the xenon−siter in air br a危w. minutes．Thereby，the oxidative ha1亡reaction started by02in air and transition state was. detectedinthiscondition．Reactioninte㎜ediatesoftheoxidativeha1f−reactionsuchasTPQi．q hadshoれ1ifetime，24butHY1（㏄cupancy1．00）stayedsti11inthesubstratechame1．Therefore，I inte叩reted−that TPQ382was not TPQ。。but TPQimq．. The pathway of provided mo1ecu1ar oxygen was proposed in ECAO and re1ated enzymes．26391n those repoれs，mo1ecu1ar oxygen was provided through the cavity which are 1ocated inβ一sandwich．These cavities inc1ude most of xenon−binding sites and correspond to. 1ow−energy pathways br oxygen entワin HPAO，PSA0，PPLO and AGAO by ca1cu1ation of切 s〃。o experiments．39However，these repoれs discussed the pathway based on the static stab1e. state such as RTO in AGA0．In contrast，Xe−RT120is the comp1ex of xenon and reaction inte㎜ediate in the oxidative ha1f−reaction．The cavity ofthe pathway此r mo1ecu1ar oxygen entry Xe−RT120in AGAO is shown in Figure VI−7．The pathway ofoxygen rcaches the active site丘。m the cavity1ocated in theβ一sandwich to Xe6site．This pathway positions in backside. ofTPQ andinthc opPositiontothe substratechame1．The distancebetweenxe6andcu was shorter than between xe6and TPQ，q by o．9A（Figure v−2o），that is，TPQ，q was血rther than cu. from Xe6．Besides，the oxidative ha1f−reaction of AGAO consists of an outer−sphere mechanism which means TPQ，q oxidized to TPQ。。at irst by o2Provided舟。m the“Potentia1. second xenon−binding site”（1ocated between TPQ。、and Tyr384in AGA0）direct1y in other previous repoれs．2526’39This site has never detect xenon atom yet，because this region consists of. hydrophi1ic side chains．Whereas，this site was u㎜easonab1e space的r mo1ecu1ar oxygen in. 一62一.

(75) 、こ〆・，遺’幽I、・・. 1も菰1丁’≡ ノ. ヘ. ．。紅. ◎一1 〆∵. ㌔・. @〕・． v −4一・ J・、一ノ’ ピ. ■ 、よ. 一一 ’ 、，，甘納，，. 一’ 一’ ㌧P ．〆＾ ■ 一 − 出 ‘1■1 ＃’ ｛『吐ギ占・」一■ 一一＝I ’ 」．. τPQ382. 細占. ，、／一. 、．！. 列. Figure Vl・9 The cavity of the pathway br mo1ecu1ar oxygen en町Xe−RT120in AGA0．The pathway of oxygen reaches the active site丘。m the cavity1ocated in theβ一sandwich to Xe6site．This. pathwaypositionsinbacksideofTPQandintheopPositiontothesubstratechame1．. AGA0because of stmctura1change舟。m“o任一。opPer”TPQ。、to‘‘on−copPer”TPQ，q．This is indicate that oxygen is provided in the“Potentia1second xenon−binding site”aier TPQ，q is. oxidizedto TPQimq orTPQ。、．Thus，mo1ecu1aroxygen inthis site is㎜concemedre−oxidationof TPQ，q．Furthe㎜ore，it was a1so suggested that the oxidative ha1トreaction in PsA0adopted the. imeトsphere e1ectron trans飴rmechanism by㎞netic studies24’46in contrast to the outer−sphere. e1ectron trans危r mechanism in HPA025and ECA0．47’48This is consistent with the putative. ca1cu1ated pathways of HPAO and other re1ated enzymes and the pathway in AGAO as a positiona1re1ationships．Consequent1y，it is presented that provided mo1ecu1ar oxygen1ocated in. xe6site oxidizes cuI to Cun at first bebre TPQ，q is re−oxidized to TPQimq using the. imer−spheree1ectrontrans危rmechanism．一63一.

(76) Conc仙s7ons I c1a㎡ied the di脆rence ofreaction time in the so1ution and in the crysta1by monitoring. the reductive ha1￡reaction in the WT AGAO crysta1s with PEA by sing1e−cワsta1 microspectrophotometry．AGAO in the crysta1s reacted with PEAduhng the time course丘。m2 to60min．Therebre，the cata1束ic reaction time ofAGAO in the crysta1s was pro1onged to as much as∼6．0x104times the reaction rate in so1ution due to the innuence ofthe protein crysta1 ie1d．. I succeeded in dete㎜ining thc stmctures of bur reaction inte㎜ediates that was the. Michae1iscomp1ex，the substrate Schi任一base，theproduct Schi任一base andAGAO佃Y1comp1ex． By ana1yzing stmcture chεmges during the reductive ha1f−reaction and by comparing those of wi1d type with those ofD298A mutant，I indicated that more precise stmctures ofthe reaction. intemediatesandthereactionmechanismwererevea1ed．Inaddition，itwasfoundthatAGAO has dif亀rent reaction rate in each subunit in the crysta1by reason of the dif胎rent subunit. stmctures which arose丘。m variation of symmetrica1mo1ecu1ar packing．There is some possibi1ity of symmetrica1mo1ecu1ar packing consisting of the recognition of stmcture di脆rence which arises to the ha1f−of−the−sitesreactivity．. Ia1so succeededtodete㎜inedstmcturesofthexenoncomp1exofAGA0／HY1comp1ex （Xe−AGA0／HY1comp1ex）．It was revea1cd that the mo1ecu1ar oxygen shou1d be provided to. Xe6positionthroughtheβ一sandwichandCuiisoxidizedbythisoxygenusingtheimer−sphere e1ectron transおrm㏄hanism． h conc1usion，thesc achievements give e1ucidation of not on1y more precise reaction. mechanismbytime−courseobsewationofstmctura1changesofreactioninte㎜ediatesbuta1so ofcata1束ic reaction which is contro11ed coordinate1y by who1e ofenzyme．. 一64一.

(77) 65一.

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(82) L7Sf OfPub〃Ca”0nS The contents of this thesis have been pub1ished or wi11be pub1ished in the曲11owing o㎡gia1papers and reports．. Papers 1. Crysta1stmctureana1ysisofreactioninte㎜ediatesofbacteha1copperamineoxidase 幽；Tominaga，A．；0tsu，M．；0k勾ima，T．；Tanizawa，K．；Yamaguchi，H．ノαo Cりノ∫吻〃。g7．2008，ノ64，C277．. 2. Detectionofthereactioninte㎜ediatescata1yzedbyacopperamineoxidase 幽；0ya，H．；Tominaga，A．；0tsu，M．；0k勾ima，T．；Tanizawa，K．；Yamaguchi， H．∫卵〃。加。炉。〃沢α冴αC．2011，ノ8，58−61．. 一70一.

(83) Repo市s 1. Time reso1ved stmcture ana1ysis ofthe enzymatic reaction in copperamine oxidase. Y乞maguchi，H．；幽；Ootsu，M．；Ito，L．∫Pκ肋g−8㏄e7亙Ψeガme〃縦ψo沌 2007．2007A1707． 2. Time reso1ved crysta11ography on the enzymatic reaction ofpheny1ethy1amine oxidase. Yamaguchi，H．；幽；0otsu，M．；Tanizawa，K．；0kajima，T．∫〃加g−8㎝ぴ亙耳ρeれme〃Rξρo以2007．2007B1394． 3. Time se㎡es cワstanographic ana1ysis of the enzymatic reaction of topaquinone containing copPer amine oxidase. Yamaguchi，H．；幽；Tominaga，A．∫P7肋g−80keκ亙Ψeれme〃。 R叩。〃，2008．. 2008A1390． 4. Time reso1ved stmctura1study on the reaction mechanism ofamine oxidase. Yamaguchi，H、；幽L；Tominaga，A．∫P7肋g−8眺ぴ亙Ψe〃me〃R印。れ，2008． 2008A6843． 5. Stmctura1Study on the enzymatic reaction ofpheny1ethy1amine oxidase. Yamaguchi，H。；幽L；Tominaga，A．∫P7肋g−8晩ぴ五Ψぴゴme〃。沢印。κ2008． 2008B1552． 6. Time reso1ved stmctura1study on the reaction mechanism ofamine oxidase. Yamaguchi，H．；幽；Tominaga，A．∫P7加g−8α∫ぴ亙Ψeれmem Rψo〃，2008． 2008B6843． 7. Stmctura1studies onthe reaction mechanism ofphenyIethy1amine oxidase. Yamag㏄hi，H．；幽L∫Pr肋g−8㏄er亙Ψeれme〃Rψo〃，2009．2009A1461．. 8. フェニルエチルアミン酸化酵素の時系列に沿った酵素反応中間体の構造解析 Y乞maguchi，H．；Kataoka M．；Oya，H．∫P7肋g−8ωer亙Ψeれme〃Rψo〃，2009．. 2009B1400．. 9. 銅・TPQ含有アミン酸化酵素の触媒反応機構の構造学的研究 Yamagu−ch1，H，Kataoka M，Oya，H∫Pη〃g−8㏄e7万Ψぴme〃。R印。〃，2009，一71一.