非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究

21卷1期生物工程学报Vol.21 No.12005年1月.Chinese Journal of BiotechnologyJanuary 2005非解朊栖热菌HG102耐热β_糖苷酶的结构与功能研究，The Structure-function Relationship of Thermostable β-glycosidase from the Thermophilic Eubacterium Thermusnonproteolyticus HG102杨雪鹏'2，杨寿钧"，韩北忠”，金城'"YANG Xue-Peng'.2，YANG Shou-Jun' , HAN Bei-Zhong?2 and JIN Cheng'" .1.中国科学院微生物研究所微生物资源前期开发国家重点实验室,北京1000802.中国农业大学食品科学与营养工程学院，北京，10001. Slate Key Laboratory of Microbial Reouces，Intite of Micobiology, Chine Academy of Sciences, Beijing 1000 China2. College of Food Science and Nuritionad Enginering. Bejing 100083 , China摘要非解朊栖热菌HG102耐热B-糖苷酶为(β/a)。桶状结构,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较，推测Glu164和Glu338分别是质子供体和亲核基团两个活性位点;在a~螺旋N端第一位的脯氨酸和蛋白质外周的精氨酸是耐热机制的关键住点和关键氨基酸残基。为确定这些氨基酸残基的功能，通过基因定点突变的方法分别把Clu164、Glu338、Pro316、Pro356 Pro344和Ang325 置换成GlnAla、Gly、Ala、Phe和Leu,同时还对Pro316和Pro356进行了双置换。突变酶经过纯化得到电泳纯,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析,结果表明Glu164 和Glu338 分别是质子供体和亲核基团,亲核基团的突变酶TnglyE338A可以合威混合型糖苷键寡糖类似物;在a-螺旋N端第一位的Pro316和Pro356 以及在蛋白质外周形成离子键的Arg325 均是对耐热性有贡献的关键氨基酸残基。关键词}$_糖苷酶， 转糖苷活性，热稳定性，定点突变中图分类号Q814文献标识码A文章编号100-3061 (2005)01 0084-08Abstract p-Clycosidase (Tngly) from the thermophilic eubacterium Thermus nonproteolyticus HG102, which is a thermostablemonomerie protein and adopts the (B/a)g barrel fold, is an excellent model system to be invetigated for the thermnostable mecha-nism, activity and substrate specificity. Here, based on the analysis of structural basis for thermostability of Tngly ( Wang et al ,2003) and comparison of other proteins structure of homofamily，Glu164 and Glu338 may act as proton donor and nucleophile inthe hydrolysis reaction respectively; proline located at N1 of a-helix and arginine which can form ion link may contribute to thethermostability. We aim to futher identifty the critical sites and the amino acid residue(s) responsible for the activity, the ther-mal stability and the substrate specifcity . Mutations had been constructed by site-directed mutagenesis. They are Glu164Gln，Clu338Ala, Pro316Gly, Arg325Leu, Pro344Phe， Pro356Ala and Pro316Gly/Pro356Ala. All mutant proteins were purified toSDS-PAGE purity 。Changes in the conformnations were examined by means of CD. The Ghu338 Ala mutant showed no detectablehydrolysis activity, but can synthesize oligosaccharides, as expected for the residue acting as the nucleophile of the reaction. TheReeived: June 17, 2004; Acepted: July 26, 2004This work was suprted by CAS Inovation Progan(No 0103).+ Crreponding autor. Tel: 86-10-62587206; E-mail: jinc@ sun. im. ac. cn中国科学院知识创新工程项目基金资助(No.0103)。杨雪鹏等:非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究8SGlu164 acts as the general acid/base catalyst in the hydrolysis reaction. Changes in stabilities of mutants compared with wild-typewere determined by means of heat inactivity experiment. These results indicate that the amino acid residue of proline that is lo-cated at N1 positions of a-helix, and Arg325 that form salt bridge between a-helices 5 and a-helices 6, are the critical sites toprotein thermostabilization.Key words B-glycosidase, transglycosylation, thermostability ，site- directed mutagenesis3_糖苷酶(E.C.3.2.1.21)生物来源广泛,可以Glu338来鉴定β_糖苷酶Tngly水解反应时的质子供水解多种β构型的糖苷键,具有广阔的应用前景"。体 和亲核基团;置换Pro316 、Arg325、Pro344和Pro356嗜热细菌Thermus nonproteolyticus HG102 的-糖苷酶来 探讨酶的耐热分子基础。Tngly基因已克隆、表达并进行了酶学性质的研1材料和方法究”。Tngly 属于糖苷酶家族1,具有葡萄糖苷酶、半乳糖苷酶、岩藻糖苷酶和甘露糖苷酶活性,在高温下1.1材料还具有转糖苷活性，它的最适水解反应温度和pH1.1.1菌株与质粒: 大肠杆菌( E. coli)AS1.1739值分别为90C和5.6,在90C时,酶的半衰期为2.5[K12r~ O(lacIPOZY) x 74]在中国科学院微生物研h。Tngly晶体结构解析表明,该酶为(B/a)。桶状结究所购买,重组质粒pHY(在pUC18载体HindI克构,分别位于第四B-sheet和第七B-sheet上的Clu164隆位点含有 β_糖苷酶目的基因)为本实验室构建。和Glu338,可能为水解反应时的质子供体和亲核基BMH71-18购自Promega 公司。团。1.1.2培养基及培养条件: LB培养基为大肠杆菌β-糖苷酶可用来合成寡糖'5.61 ,但反应要在有机完全培养基，固体培养基加入1.5%的琼脂粉。重相中并需要高浓度的糖基供体。Mackenzie 等人将组菌培养时加入氨苄青霉素(100 pg/mL),培养温度Agrobacterium sp. β-葡 萄糖苷酶的亲核基团谷氨酸为37C ;诱导时液体培养基加入1%( WIV)乳糖,固残基突变成甘氨酸，使得突变的酶只能合成糖苷键，体培养基涂布4山IPTG(200 mg/mL)40 μl X-gal(20不再具有水解功能，从而能使寡糖的产率达到mg/mL)。90%”。R_糖苷酶Tngly 可以在65C下水解乳糖或1.1.3酶及生化试剂: 定点突变试剂盒GeneEdi-纤维二糖生成三糖}21,说明酶的活性中心适合转糖tor"M in vitro Site- Directed Mutagenesis System Kit购自苷反应，可用来合成寡糖,置换耐热β_糖苷酶的亲核Promega公司; T4 Polynucleotide Kinase 购自Promega基团来合成寡糖更具有优势。已知结构的蛋白质大公司;溶菌酶购自华美公司;CMP-3-Fluoro- neuraminic约10%为(β/a)。桶状结构,或叫TIM结构)，因此以Acid购自Calbiochem-Novabiochem 公司;胰化蛋白胨Tngly作为模型来研究(β/a)。桶状结构的稳定机制非(TRYPTONE)和酵母提取物(YEAST EXTRACT)购自常有意义。文献报道，在a-helixN端第一位脯氨酸OXOID公司;IPTG、X-gal .ONPG和乳糖购自Sigma公的刚性结构和蛋白质外周精氨酸形成的离子键可能司;蛋白质分子量标准购自华美公司;其它试剂均为对蛋白质热稳定性有一定的贡献”。分析纯试剂。本实验在Tngly晶体结构的基础上,用基因定所用诱变寡核苷酸为上海生工公司合成，见点突变的方法置换Tngly的氨基酸残基Glu164和表1。表1置换氨基酸位点及其在蛋白质二级结构中的位置和相应的诱变寡核苷酸设计Table 1 Oligonucleotides and mutagenic position in proteinMutationPosition in proteinMutagentic olgonucleotidesGlul64ClnNo.4,B-sheet5'-ACCCTGAACCAGCCCTCCTGC-3'Glu338AlaNo.7, B-sheetS-TACTCGCCCCCCC-3'Pro316GlyNo.6, a-helix NI5'-GGGAGGTCTACGGCGACGCGCCTT-3'Anrg325Leulon link5-CTCTTCAAGCTCCCGCCGG-3'Po344PheNo.7, B-shet5-CCCCCCCTACTTCGACCTCTGGAC-3'Pro356AlaNo.7, a-helix NI5'-GTGGAGGACGCCGACGGGTG-3'Pro316Gly/No.6, a-helix N1/5'-GCGAGCTCTACGGCGACCCGCTT-3'5'-GTGGAGGACGCCGACCCGGTC-3'Bold and underdined mucleotides are the mutations sites.8Chinse Joumal of Biotehnology生 物工程学报2005, Vol.21 ,No.11.2 方法SWISS MODEL. html网站上完成8910)。1.2.1基因定点突变: 以重组质粒pHY单链DNA1.2.6转糖苷反应:突变酶TnglyE338A和各种底物为模板,在诱变寡核苷酸介导下,用定点突变试剂盒在65C,pH6.8下反应2h,薄层层析检测。相应的试剂进行突变和筛选。筛选出单菌株提取质1.2.7薄层 层析(TLC):展开剂为正丁醇:乙酸:水粒送交TaKaRa Biotechnology (Dalian)测序鉴定突变=1:2:1;显色剂为苯胺:二苯胺:磷酸=5:5:1(显色结果。范围为10 ug)。1.2.2突变基因 的表达和蛋白质纯化:用突变的在数据的测定当中都进行3次或3次以上试重组质粒转化到大肠杆菌(E. coli)AS1 .1739,从过验 ,误差范围在5%以内。夜培养的Amp-LB平皿上挑取单菌落,接种到5 m[2结果液体LB(Amp 100 pg/mL)培养12 h,1%接种量接种到100 mL Amp-LB液体培养基中培养12 h;再以1%2.1基因定点突变构建突变酶基因的接种量接种到4 L Amp-LB液体培养基(5 L发酵以单链重组质粒为模板，由诱变寡核苷酸和选罐)中培养,加入1%的乳糖诱导, 37C ,300 r/min的择寡核苷酸介导突变和筛选突变基因"。突变质搅拌速度,通无菌空气,培养28 h。粒与野生质粒大小相同。测序结果显示突变质粒收集发酵罐中的4 L发酵液(4C, 6000g, 15pHYE164Q编码164位上的Clu碱基密码子GAG突min)离心,菌体用磷酸缓冲液(50mmol/L,pH6.6)悬变为CIn的密码子CAG;突变质粒pHYE338A编码浮;冰浴超声破碎,离心去除细胞碎片;在80C水浴338位上的Glu碱基密码子GAA突变为Ala的密码恒温加热15 min,离心(4C , 10000g, 15 min)取上清。子GCA;突变质粒pHYP316G编码316位上的Pro碱向上清液中缓慢加入固体硫酸铵,收集30% ~ 60%基密码子CCC突变为Gly的密码子GGC;突变质粒饱和度的沉淀,溶于磷酸缓冲液中(50mmol/L,.pHYP356A编码356位上的Pro碱基密码子CCC突pH6.6),用同种缓冲液透析过夜。除盐的粗酶冻干变为Ala的密码子GCC;突变质粒pHYP344F编码浓缩后,在AKTAFPLC蛋白质纯化系统上用DEAE344位上的Pro碱基密码子CCC突变为Phe的密码离子交换柱进行纯化，缓冲液A为磷酸缓冲液(50子CTT;双突变质粒pHYP316G/P356A编码316位和mmol/L,pH6.6),缓冲液B为1 mol/L NaCl溶于磷酸356位上的Pro碱基密码子CCC分别突变为Gly的缓冲液(50 mmol/L, pH6.6),洗脱条件为在5个柱体密码子CGC和Ala的密码子GCC; 突变质粒积内B溶液比例上升到30%，测酶活和SDS-PAGEpHYR325L编码325位上的Arg碱基密码子CGC突.检测目的蛋白纯度,收集合并酶活峰,用冻干机冻干变为Leu的密码子CTC。浓缩,用蒸馏水溶解冻干的酶蛋白，在磷酸缓冲液2.2突变基因的表达和突变酶的纯化(50 mmol/L, pH6.6)中透析,再用Superdex G-75分子含有突变基因的质粒分别转化到大肠杆菌筛柱层析纯化,洗脱液为磷酸缓冲液(50mmol/L,AS1.1739中,经乳糖诱导,发酵罐大量培养,目的基pH6.6)。以上步骤均在常温下进行。因得到大量表达,将表达产物分别经加热分离、硫酸1.2.3酶活测定: 0.1 mL 4 mmol/L ONPC,0.1 mL铵分级沉淀、DEAE和SuperdexG75分子筛层析纯pH5.8磷酸缓冲液,0.7 mL H20,混匀后于85C水浴化,得到电泳纯，纯度均达90%以上(图1);除保温5 min,加入0.1 mL酶液,反应10 min,加入4TnglyE164Q和TnglyE338A外,其他突变酶在在85CmL Na,CO,溶液终止反应,420 nm测定吸光值。此时的比活均与野生酶相当(表2)。条件下每分钟产生1 pmol对硝基苯酚的酶量为一2.3突变酶与野生 酶结构的比较个酶活单位。野生和突变纯酶由CD光谱仪直接扫描得到观1.2.4蛋白含量测定:以牛 血清白蛋白为标准，察值(mdeg)，把观察值换算成平均残基椭圆度(meanLowry 法测定。residuellplicit)。结果显示突变酶与野生酶结构1.2.5 CD 光谱分析和同源建模: - .定浓度的各种相同，如图2(因为CD光谱图曲线拥挤,这里只显示酶蛋白分别加入测量杯中，把测量杯放人光谱仪两个突变酶和野生酶的CD光谱图)。说明单个氨(JASC0 J-715)中,用200 ~ 250nm的光进行扫描。用基酸残基的置换没有改变蛋白质的折叠。同源建模蛋白质分子量和氨基酸残基数目计算残基椭圆度。结果也显示置换位点的结构域与野生酶相同(见图同源建模在ht://ww. expasy . org/wissmod/3),这与CD光谱结果一致。杨雪鹏等:非解朊栖热菌HG102酎热β糟苷酶的结构与功能研究.7表2 I 突变纯酶的比活Table 2 The specific activity of purifted TnglysWild-TnglyPurified proteinstype_E338AE1640P316GP356AP344F P316G/P356AR325LSpecifie activity/( u/mng)17.600.9820.021.017.924.010005000......-- - TnglyP316G/p356A粤-500020一喜-00000.-15000图1 SDS-PAGE 分析野生酶Tngly和突变酶的纯化-20000 :Fig.1 SDS- PAGE pattern of purifed Tnglys2122232402501:Tnpgly E164Q; 2:Tngly P344F; 3:Tngly P316G;入num4:Tngly P356A; S:Tngly R325L; 6:Tngly P316G/P356A;图2突变酶和野生酶 CD光谱图.7; wild-type; Mstanderd marker proleins.Fig. 2 1 Cireular dichroie speclra of wild-type Tnglyand two mutant TnglyR325L, TnglyP316C/P356A in the2.4质子供体 与亲核基团的确定far-ultraviolet region突变酶TnglyE164Q的比活是野生酶比活的The secta were measured on a JASCO J-715 setrolarineter at 25C.5.5%，且几乎不受反应液pH值的影响(见图4);突The protein concentration of wild-type Tngly was 0. 166mng/mL, the Tng-变酶TnglyE338A检测不到水解活性,在CMP-3-F-lyR325L was 0.053mn/ mL, the TngyP316G/P356A was 0.079mg/mL alNeu5Ac和甘露糖为底物的情况下表现出转糖苷功pH6.6, 50 mmol/ mol phosporie acid bulfr.能(见图5),用相应的糖苷酶Tngly和a-2-(3, 6)-和底物形成共价复合物,是双置换反应的关键步骤。NANase II都没能把反应产物水解。根据以上结果突变酶TnglyE338A失去水解功能而只有转糖苷功确定Glu164为水解反应时的质子供体,介导水的攻能,可作为糖苷合成酶用于寡糖的合成。击，Glu338为水解反应时的亲核基团,其侧链羧基345。 3434356443575435T 34626 !2S324359203231932215D图3突变点的结构和野生酶的比较立体图Fig. 3 Stereo drawings showing the mutant structure in the vicinity of the mutant sitesWild- type and mutants stutures are superimposed. A:Ar325L; B:P344F; C:P316G; D:P356A88Chinese Jourmal of Biotechnology生物工程学报 2005, Vol.21,No, I .0.8 .Wild-type Tngly0.5-←wT一- TnglyP344F...... TnglyR325L一TnglyE164Qa6-+- Tgl1g6P3360.4-TnglyP316GTnglyP356Ag0.2+0.1-0.20.0-0.0070809100图4突变酶TnglyE164Q和野生酶的活性受反应液pH值Temperature/C的影响图6突变酶的最适反应温度Fig. 4 Comparison of the pH dependence on the ONPG substrateFig. 6 Elects of temperature on ste-diretedwild-type Tngly and TnglyE164Q at the same condition of reactionmutagenesis Tngly hydrolysis activitiesThe hyddrolysis activity was determined as descnibedin Materials and Mcthods at diferent tempernturesfrom s0C to 95C.0.7-.. **. TnglyP344F0.6-TnglyR325L●。0.51Tngly316G/P356A-Highigtnder3+ulravioletradiation0.2个图5突变酶TnglyE338A的转糖昔反应TLC图谱Fig. 5 Transglycosylation activity of mutant0.0+TnglyE338A at 65C ,pH6.8pF1:mannose; 2:sialic acid; 3:CMP-3-F-Neu5Ac; 4: mannose and CMP-3-F-Neu5Ac reacted with mutant TnglyE338A; 5: mannose and CMP-3-F.图7突变酶的最适反应pHNeu5Ac with no enzyme; 6: production of reaction of lane4 reacted withFig. 7 Elects of pH on sile-directed mutagenesiswild-type Tngly; 7: production of reaction of lane4 reacted with a-2-(3,Tngly hydrolysis activities6)-NANase I (raction of condition rely on the enxyme of opimum con-The hydrolysis activity was determined as dernbeddition) .in Materials and Methods at diferet pH from 4.5 to 9.0.2.5热稳定 性相关的突变酶酶学性质分析2.5.2最适pH值:如图7所示,6个突变酶的最适2.5.1最适温度:在50~95C的温度范围内测定.pH值都是大约pH5.8,与野生酶相似。说明置换的野生酶和突变酶的最适反应温度,发现突变酶.氨基酸不影响酶活性中心的环境,也表明置换的点TnglyP344F的最适反应温度与野生酶都为90C,其在蛋白的外周不影响酶蛋白质的活性。它突变酶最适反应温度都有所下降(如图6),2.5.3热稳定性: 突变酶和野生酶在85C温浴不TnglyP316C和TnglyP3S6A 最适反应温度大约为同的时间，然后测剩余酶活,当剩余酶活是原来的879 ,Tngly-P316G/P356A和TnglyR325L最适反应温50%时,各种酶经历的时间(1n2)分别是野生酶Tngly度大约为85C。结果表明,位于第六a-helix的N端63 min,突变酶TngyP344F 57 min , TnglyP316G 43 min,第一位的P316、位于第七a-helix的N端第一位的TnglyP356A 45 min,TnglyR325L 33 min和TnglyP316G/P356和位于第六a-helix的R325置换都降低了酶蛋P356A 15 min(见图8)。把各种酶在不同的温度温白的刚性结构，但在Loop区的P344对酶的结构刚浴15min测定酶的剩余酶活,得出相似的结果,Tm性影响较小。值是酶剩余50%活力时所对应的温度如图9,结果杨雪鹏等:非解朊栖热菌HG102耐热β_糖苷酶的结构与功能研究89110 1力学常数,底物浓度由2mmol/L到40mmol/L。结果- Wild-type100 '. Pro316Gly/Pro356Ala显示动力学常数基本相同(见表3),表明突变酶置)0一. Pro356Ala30Pro316Gty换的氨基酸不影响酶的亲和性。Ang325Leu. Pro344Phe60 t表3野生酶及其突变酶 热稳定性和反应动力学常数50 |Table 3 Thermodynamic and kinetic Parameters forwild-type Tngly and its mutants01Protetin'K.(kem/K)/m19(1(mmol/L) /[U(mol-s)]Wild-ype 63+0.5 94.0+0.5.377.90.010.0 20.0 30.0 40.0 50.0 60.0 700/minPro316G]y 43+0.5 91.0+0.5 -3.0+0.5 1.277.1图8野生酶和突变酶的热稳定性分析Pro356Ala 45+0.5 91.5+0.5 -2.5+0.5 1.277.2Fig. 8 Kinetics of inctvivation of wild-type TnglyPro344Phe 57+0.5 93.0+0.5 -1.0+0.5 1.378.1and its mutants at 85C Values whose error ranges wereArg325leu 33+0.5 89.0+0.5 -5.0+0.578.6within 5% were averagedPro316Cly/15+0.5 86.5土0.5 -7.5+0.5.478.211Pr356Ala100a: Determined by kinetics of irevernible heat inetivity at 85C.b: Mecling temperature from Fig.9.)te: btained by catalyie reaction of enzyme at optimun temperature.专603讨论曾5+Wlild-ype糖苷酶家族1 β_糖苷酶采用保留型双置换催化营40 +◆Pro316Gly.机制,在酶的活性部位存在两个重要的羧酸部分,一正3甘Pro316Gly20 →Pro356Ala个质子化,称为质子供体,另一个发生离子化,称为. Ang325Leu亲核基团,所切糖苷键的氧原子被质子化的羧基攻r t Pro34PheoL击，糖苷键断裂,形成的碳正离子与离子化的另-一个75.0 80.085.010.0Incubation temperature/C羧基以离子键或共价键的形式形成中间产物，此中图9野生酶和突变酶的热稳定性分析间产物不稳定，当亲核试剂进行亲核攻击时,此中间Fig. 9 Efect of temperature on the stability of产物不存在,取而代之的是一个新的糖苷键 ,当亲核试剂是水时,发生水解反应,当亲核试剂是醇或某个Each purtied enyme was teated t difret teperntures for 15 miutes.糖的羟基时，则发生转糖苷反应8.121。质子供体在The remaining activities were expressed 越percentages of the original ac-双置换当中起到酸/碱催化的双功能,在第-.步提供tivities. Values whose error ranges were within 5% wene avenged.质子,在第二步介导水的攻击明,亲核基团是形成显示野生酶和突变酶TnglyP344F 、Tngly-P316G、酶与底物中间物的关键氨基酸残基。TngIyP356A .TnglyR325L、Tng!yP316G/P356A的T.值Agrabacterium faecalis β-葡萄糖苷酶的质子供体分别是94. .93.91.91、89和86C。以上结果表明位和Sufolobus solfataricus β_糖苷酶两个活性位点的谷于第六a-helix 的N端第一位的P316和位于第七氨酸残基都已确定[15.16)。目前,在糖苷酶家族1中a-helix的N端第-.位的P356刚性结构及位于第六除了葡萄糖硫苷酶外，质子供体和亲核基团两个活a-helix的R325所形成的离子键对酶蛋白的热稳定性位点都是谷氨酸,分别在第四B-sheet 和第七B-性有一定的贡献，而在Loop区的P344对蛋白质的sheet.上。这两个活性位点氨基酸残基在家族1中很.稳定性影响较小。保守,靠近蛋白质N端是T(F/LM)NE(P/L/I) ,靠近2.5.4突变酶米氏常 数(K )和转化数(..)的测C端是(IV)TENG。在Tngly结构当中,E164所在定:野生酶及突变酶可以水解ONPCle、ONPGal、-TLNEP-区 域和E338所在ITENG-区域，分别位于第ONP-GFuc . PNPGle、PNPGal、PNPFuc和PNPMan等多四B-sheet和第七B-sheet 上,可推测E164和E338可种底物,这里只选择ONPGIe作为底物来测定酶的动能为R-_糖苷酶Tngly的两个活性位点，在水解反应当9Chinese Journal of Biotechnology 生 物工程学报2005, Vol.21,No.1中前者作为质子供体，后者作为亲核基团。Glu164P344的B值分别为25.27.28.51和29.97。B值和.和Clu338分别置换为GIn和Ala的突变试验结论与原子的稳定性有关,B值越小原子群的稳定性越大。上述推测- -致。β-糖苷酶Tngly热稳定性可能不只是含有脯氨糖苷酶水解反应时糖苷键的水解有立体选择酸的结果,在蛋白质- .级序列当中含有9.6%的精性，但合成糖苷键时,合成的键型是多样的,可能是氨酸,在3D结构当中发现精氨酸大部分位于蛋白β键或a键,也可能是1-3、1-4或1-6等[”。耐热突质外周,在a-helix之间形成离子键网络。这样的离变酶TnglyE338A在65C温度下可催化底物CMP-3-子键在其他耐热蛋白质中都被证明为重要的稳定因F-Neu5AC和甘露糖的合成，合成产物不能用相应的30011231。较早Penutz等人比较了同一种酶不同糖苷酶Tngly和a-2-(3,6)-NANase I水解,表明产物来源的耐热酶和常温酶的结构发现,耐热酶的分子的糖苷键键型可能发生了改变。这样产生的混合键表面离子键比常温酶的多0。在以后的耐热酶研型连接的寡糖(mixed-linkage)有可能作为寡糖类似究中也发现类似的情况，比如,苹果酸脱氢酶{4、甘物用于糖苷水解酶的抑制剂[8。油醛-3-磷酸~脱氢酶'35]和DNA聚合酶[6]等。我们蛋白质的耐热因素,包括离子键作用、氢键作把6-a-helix的Arg325 和5-a-helix的Asp235 之间形用疏水作用、金属键、二硫键、包装效应、Proline理成的离子键打断,突变酶R325L的热稳定性参数r。论、a-螺旋的稳定作用和氨基酸组成等都经过深入值下降大约5C,表明蛋白质外周的离子键对野生广泛的分析(9,20.21 ,从大量的研究中发现,一个蛋白酶Tngly热稳定性也有贡献。质的耐热机制可能是多种因素的结果。各个突变酶与野生酶纯化方法相同,从SDS-Proline 与其他的a-氨基酸不同，由于其N原子PAGE电泳结果分析各个突变酶与野生酶纯度相位于吡略环上,使得前一个氨基酸与它形成肽键时当,然而突变酶TnglyP316G、TnglyP356A、TnglyP316G/(C*-N)不能自由旋转,另外吡咯环还具有疏水作P356A和TnglyR325L的比活与野生酶相比有所上用。Proline 分子的这些特点,在形成肽链时导致它升,可能的原因是在85C时突变酶结构与野生酶相比其它氨基酸的构型熵小2.20),从而降低蛋白质比“刚性”下降“柔韧性”增加,有利于突变酶催化反的折叠熵。蛋白质的折叠熵降低可以提高其稳定应。突变酶TnglyP316G、TnglyP356A、TnglyP316G/性5.26。较早,从统计的结果表明蛋白质中脯氨酸P356A、TnglyP344F和TnglyR325L动力学常数与野生含量增多,可以明显提高蛋白质的热稳定性田,但酶基本相同,说明被置换的氨基酸残基与酶的活性脯氨酸在蛋白质二级结构中的位置不同对稳定性的无关,置换上来的氨基酸残基也不影响酶活性中心贡献不一样,动力学模拟试验证明脯氨酸位于ar结构的正确折叠。helix的N端第一位最有利于蛋白质的稳定性。统计结果也表明脯氨酸偏向位于a-helix的N端第REFERENCES(参考文献)-.位和B-tum的第二位9],这可能有利于蛋白质的[1] IehikawaY, Look cC, Wong CH. 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