Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments

We prepared two dissected fragments of hen lysozyme and examinedwhether or not these two fragments associated to form a native-likestructure. One (Fragment I) is the peptide fragment Asn59–homoserine-105containing Cys64–Cys80 and Cys76–Cys94. The other(Fragment II) is the peptide fragment Lys1–homoserine-58connected by two disulfide bridges, Cys6–Cys127 and Cys30–Cys115,to the peptide fragment Asn106–Leu129. It was found thatthe Fragment I immobilized in the cuvette formed an equimolarcomplex with Fragment II (K_d = 3.3x10^–4 M at pH 8 and25°C) by means of surface plasmon resonance. Moreover, fromanalyses by circular dichroism spectroscopy and ion-exchangechromatography of the mixture of Fragments I and II at pH 8under non-reducing conditions, it was suggested that these fragmentsassociated to give the native-like structure. However, the mutantFragment I in which Cys64–Cys80 and Cys76–Cys94are lacking owing to the mutation of Cys to Ala, or the mutantfragment in which Trp62 is mutated to Gly, did not form thenative-like species with Fragment II, because the mutant FragmentI derived from mutant lysozymes had no local conformation dueto mutations. Considering our previous results where the preferentialoxidation of two inside disulfide bonds, Cys64–Cys80 andCys76–Cys94, occurred in the refolding of the fully reducedFragment I, we suggest that the peptide region correspondingto Fragment I is an initiation site for hen lysozyme folding.     

The double catalytic triad, Cys25-Hisl59-Aspl58 and Cys25-Hisl59-Asnl75, in papain catalysis: role of Aspl58 and Asnl75

The 1.65 Å X-ray structure of papain, which exhibits aCys25-Hisl59-Asnl75 triad, does not correspond to the catalyticallyactive ion pair state since Cys25 is oxidized to cystelc acidand Hisl59 is predominantly neutral. Thus, stochastic boundarymolecular dynamics simulations starting from the 1.65 ÅX-ray structure of papain have been performed for Cys25 andHisl59 in the SH-ImH⁺, SH-Im, S^-ImH⁺ and S^--Im states and forAspl58 mutated to Asn, Glu and Gly in the ion pair state. Bycomparing the resulting averaged structures and analyzing thetrajectories of certain interatomic distances, important differencesin the active-site geometry of papain have been found. In particular,the initial Cys25(S^-)-Hisl59(ImH⁺)-Asnl75(C=O) triad found inthe X-ray structure is retained in all the structures exceptthe wild type and Aspl58 - Asn ion pair states where there isa confonmational transition to form the triad, Cys25(S^-)-Hisl59(ImH⁺)-Aspl58(COO^-). Both triads, Cys25(S^-)-Hisl59(ImH⁺)-Aspl58(COO^-)and Cys25(S^-)-Hisl59(ImH⁺)-Asnl75(C=O) are postulated to participatein catalysis and their roles are discussed. Thus, catalysisdoes not take place from a single sterk position but a two-statemechanism     

The dipeptide H-Trp-Glu-OH shows highly antagonistic activity against PPARgamma: bioassay with molecular modeling simulation

The peroxisome proliferator-activated receptor gamma (PPARgamma) is an important therapeutic drug target for several conditions, including diabetes, inflammation, dyslipidemia, hypertension, and cancer. It is shown that an antagonist or partial agonist of PPARgamma has attractive potential applications in the discovery of novel antidiabetic agents that may retain efficacious insulin-sensitizing properties and minimize potential side effects. In this work, the dipeptide H-Trp-Glu-OH (G3335) was discovered to be a novel PPARgamma antagonist. Biacore 3000 results based on the surface plasmon resonance (SPR) technique showed that G3335 exhibits a highly specific binding affinity against PPARgamma (K(D) = 8.34 microM) and is able to block rosiglitazone, a potent PPARgamma agonist, in the stimulation of the interaction between the PPARgamma ligand-binding domain (LBD) and RXRalpha-LBD. Yeast two-hybrid assays demonstrated that G3335 exhibits strong antagonistic activity (IC50 = 8.67 microM) in perturbing rosiglitazone in the promotion of the PPARgamma-LBD-CBP interaction. Moreover, in transactivation assays, G3335 was further confirmed as an antagonist of PPARgamma in that G3335 could competitively bind to PPARgamma against 0.1 microM rosiglitazone to repress reporter-gene expression with an IC50 value of 31.9 muM. In addition, homology modeling and molecular-docking analyses were performed to investigate the binding mode of PPARgamma-LBD with G3335 at the atomic level. The results suggested that residues Cys285, Arg288, Ser289, and His449 in PPARgamma play vital roles in PPARgamma-LBD-G3335 binding. The significance of Cys285 for PPARgamma-LBD-G3335 interaction was further demonstrated by PPARgamma point mutation (PPARgamma-LBD-Cys285Ala). It is hoped our current work will provide a powerful approach for the discovery of PPARgamma antagonists, and that G3335 might be developed as a possible lead compound in diabetes research.     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca²⁺-binding protein recoverincontains four potential EF-hand Ca²⁺-binding sites, of whichonly two, the second and the third, are capable of binding calciumions. We have studied the effects of substitutions in the second,third and fourth EF-hand sites of recoverin on its Ca²⁺-bindingproperties and some other characteristics, using intrinsic fluorescence,circular dichroism spectroscopy and differential scanning microcalorimetry.The interaction of the two operating binding sites of wild-typerecoverin with calcium increases the protein's thermal stability,but makes the environment around the tryptophan residues moreflexible. The amino acid substitution in the EF-hand 3 (E121Q)totally abolishes the high calcium affinity of recoverin, whilethe mutation in the EF-hand 2 (E85Q) causes only a moderatedecrease in calcium binding. Based on this evidence, we suggestthat the binding of calcium ions to recoverin is a sequentialprocess with the EF-hand 3 being filled first. Estimation ofCa²⁺-binding constants according to the sequential binding schemegave the values 3.7 x 10⁶ and 3.1 x 10⁵ M^–1 for thirdand second EF-hands, respectively. The substitutions in theEF-hand 2 or 3 (or in both the sites simultaneously) do notdisturb significantly either tertiary or secondary structureof the apo-protein. Amino acid substitutions, which have beendesigned to restore the calcium affinity of the EF-hand 4 (G160D,K161E, K162N, D165G and K166Q), increase the calcium capacityand affinity of recoverin but also perturb the protein structureand decrease the thermostability of its apo-form.     

Free energy perturbation study on a Trp-binding mutant (Ser88 ->Cys) of the trp-repressor

The Ser⁸⁸Cys mutant of the trp-repressor showed a lower affinityfor the corepressor than the wild-type repressor [G = 1.7 ±0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264,18314–18319].A molecular dynamics/free energy cycle perturbation study wasperformed to understand the origin of the decreased affinity.A value (G = 1.58 ± 0.28 kcal/mol) comparable with theexperimental value was obtained by the simulation. Free energycomponent analysis revealed that destabilization of the vander Waals interaction between Ser⁸⁸ and Trp¹⁰⁹ (corepressor)mainly contributed to the decreased affinity of the mutant.The rotational transition of the hydroxyl (sulfhydryl) groupof Ser⁸⁸ (Cys⁸⁸) during the simulations affected the contributionsof Arg⁸⁴ and water to the free energy change in the aporepressorand those of Arg⁸⁴ and Trp ¹⁰⁹ to that in the holorepressor.However, the contributions from different residues compensatedeach other, and the total free energy changes were almost invariablein the various simulations.     

Homology modeling of a heme protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase

A 3-dimensional model of lignin peroxidase (LiP) was constructedbased on its sequence homology with other peroxidases, particularlycytochrome c peroxidase, the only protein with a known crystalstructure in the peroxidase family. The construction of initialconformations of insertions and deletions was assisted by secondarystructure predictions, amphipathic helix predictions, and considerationof the specific protein environment. A succession of moleculardynamics simulations of these regions with surrounding residuesas constraints were carried out to relax the bond lengths andangles. Full protein molecular dynamics simulations with explicitconsideration of bound waters were performed to relax the geometryand to identify dynamically flexible regions of the successivemodels for further refinement. Among the important functionallyrelevant structural features predicted are: (i) four disulfidebonds are predicted to be formed between Cys3 and Cys15, Cys14and Cys285, Cys34 and Cys120 and Cys249 and Cys317; (ii) a glycosyla-tionsite, Asn257, was located on the surface; (iii) Glu40 was predictedto form a salt bridge with Arg43 on the distal side of the hemeand was considered as a possible origin for the pH dependenceof compound I formation; and (iv) two candidate substrate bindingsites with a cluster of surface aromatic residues and flexiblebackbones were found in the refined model, consistent with thenature of known substrates of LiP. Based on these predictedstructural features of the model, further theoretical and experimentalstudies are proposed to continue to elucidate the structureand function of LiP.     

高级氧化技术矿化水中有毒有机物理论极限能耗

通过过程耦合的概念建立了高级氧化技术矿化有机污染物理论极限能耗的计算方法,并分析了通过与高级氧化技术中4种可能的氧化剂矿化反应分别去除1000kg19种代表性有机污染物(8种氯代烷烃、4种氯代烯烃、3种溴代甲烷、4种芳香烃及其衍生物)的理论极限能耗,将计算结果与物理法脱除有机污染物的理论能耗进行了比较分析。研究结果表明,高级氧化技术矿化水中有机污染物为放热过程,且高级氧化技术矿化水中有机污染物理论极限能耗较物理法脱除有机污染物极限能耗有高出数量级的差别。矿化氯代烷烃、氯代烯烃和溴代烷烃的理论极限能耗随着有机物中C—Cl或C—Br取代基的增加而普遍降低,且矿化氯代烷烃的理论极限能耗大小为氯代甲烷氯代乙烷氯代丙烷,该结论与物理法脱除有机污染物理论能耗规律一致。此外,不同氧化剂矿化氯甲烷的理论极限能耗为O3O-.OHO2;而对其他研究体系而言,不同氧化剂矿化的理论极限能耗为O-O3.OHO2。     

重组柠檬酸杆菌合成紫色杆菌素的工艺条件优化

以L-色氨酸为前体物,对弗氏柠檬酸杆菌(Citrobacter freundii)基因工程菌株生产紫色杆菌素的工艺条件进行了优化。通过单因素实验研究了培养基种类、培养温度、碳源、溶氧、种子液的状态、接种量、诱导时机、诱导剂的浓度及初始pH对细胞生长和紫色杆菌素产量的影响,通过正交实验研究了这些因素中可能存在交互作用的4个因素(种子液OD660、接种量、诱导时机、诱导剂浓度)的影响主次,确定了这些因素、水平的最佳搭配方案。结果表明,重组柠檬酸杆菌合成紫色杆菌素的最优培养条件为:种子液培养至OD660=3.6时,以3%的接种量接种于以甘油为碳源、初始pH为6.5的磷酸盐发酵培养基E2(25μg.ml-1卡那霉素),先在37℃、200r.min-1下培养至OD660=1.4,然后加入0.5μl.ml-1的诱导剂正辛烷,同时转入20℃,在150r.min-1下诱导培养31h。在此最优条件下,紫色杆菌素粗提物(紫色杆菌素及脱氧紫色杆菌素的混合物)产量可达1.809g.L-1,比优化前(0.514g.L-1)提高了252%,是目前国际上其他研究小组报道的最高摇瓶产量(0.43g.L-1)的4.2倍。质粒稳定性实验结果表明重组柠檬酸杆菌在抗生素选择压力条件下具有良好的遗传稳定性。     

Locating the unpaired cysteine of tissue-type plasminogen activator

Variants of tissue-type plasminogen activator (t-PA) were constructedwith selected cysteines replaced by alanine to evaluate therole of an unpaired cysteine, which has been presumed to bein the growth factor module. C75A, C83A, C84A and CC83–84AAvariants of t-PA were expressed transiently in human embryonickidney cells. The biochemical properties of these variants providedexperimental evidence to identify the unpaired cysteine in t-PA.Assays of amidolytic activity, plasminogen activation (in thepresence or absence of fibrinogen or fibrin), plasma clot lysis,fibrin binding, clearance in mice, and interaction with a panelof monoclonal antibodies were performed as the basis for comparingthese variants with wild-type t-PA. In all assays, C83A t-PAwas biochemically equivalent to wild-type t-PA. C75A t-PA, C84At-PA and CC83-84AA t-PA variants exhibited reduced activitiesin a variety of functional assays. These variants displayedtwo- to threefold lower activity in fibrinogen or fibrin stimulatedplasminogen activation, and fivefold reduced plasma clot lysisactivity compared with that of wild-type t-PA. The affinityof C75A t-PA and C84A t-PA for fibrin was decreased more thantwo orders of magnitude compared with C83A t-PA or wild-typet-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced2-fold in mice. The C75A, C84A and CC83–84AA variantsdisplayed significantly decreased reactivity with anti-tPA monoclonalantibodies specific for finger/growth factor domain epitopes.These data are consistent with a disulfide linkage of Cys75with Cys84 and that Cys83 exists as an unpaired sulfhydryl.The significance of the unpaired cysteine is as yet undeterminedsince C83A t-PA and wild-type t-PA are functionally equivalent.     

The role of the Cys191-Cys220 disulfide bond in trypsin: new targets for engineering substrate specificity

The S1 binding site of trypsin is cross-linked by the conserved Cys191- Cys220 disulfide bond. The substitution of Cys191 and Cys220 with Ala decreases the activity of trypsin by 20-200-fold as measured by kcat/K(m) for the hydrolysis of amide substrates; in contrast, ester hydrolysis is decreased by < 10-fold. Similar decreases are observed in the hydrolysis of oligopeptide and single amino acid substrates. This decrease in activity results from a decrease in the acylation rate. The substrate binding and deacylation rate are not affected by the loss of the disulfide bond. C191A/C220A binds BPTI with the same affinity as trypsin, although the affinity of benzamidine is decreased 10-fold and the affinity of leupeptin is decreased 1000-fold. The CD spectrum of C191A/C220A displays significant differences from that of trypsin; these differences most likely result from the loss of the disulfide chromophore, although perturbation of enzyme structure cannot be discounted. The loss of the Cys191-Cys220 disulfide has no effect on the stability of trypsin as measured by urea denaturation. Single and double substitutions of Ser at positions 191 and 220 have a similar activity to C191A/C220A. These results indicate that the Cys191-Cys220 disulfide bond is not essential for the function, structure or stability of trypsin.      

A calmodulin-target peptide hybrid molecule with unique calcium-binding properties

This paper describes the production and properties of a hybridprotein comprising the full length of the Xenopus laevis cabnodulin(CaM) sequence, followed, through a gh/cylgh/dne linker, bythe 26-residue CaM-binding region of myosin light-chain kinase(M13). This hybrid molecule appears to have high thermal stability(T_m > 75°C in the presence of Ca²⁺) as well as unusualCa²⁺-binding properties: (i) a wide-range biphasic Ca²⁺-bindingresponse (extending over pCa 4.8-7.4) and (ii) a high apparentbinding constant (pCa_50% = 6.3, a 10-fold increase from thatof wild-type CaM). NMR and CD data indicate that the CaM-M13hybrid molecule exists in equilibrium in an approximate 1:1ratio between two major conformations, one of which is similarto the compact globular structure of the CaM-M13 complex [M.Daira,G.M.Clore, A.M.Gronenborn, G.Zhu, C.B.Klee and A.Bax (1992)Science, 256, 632-638] and the other to the dumbbell-like structureof the wild type CaM [Y.S.Babu, C.E.Bugg and W.J.Cook (1988)J. Mol. Biol., 204, 191-204]. The biphasic Ca²⁺-binding curvecan be interpreted using a linear combination of two Hill bindingcurves with significantly different dissociation constants (2x 10^-6 M and 8 x 10^-6 M), which can be attributed to the twoconformations in equilibrium. The present study has opened anavenue to engineer proteins with higher Ca²⁺-binding affinitiesusing the known CaM structures as a template Received April 23, 1993; revised August 5, 1993; accepted August 25, 1993.     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine

The Staphylococcus hyicus lipase is an acyl hydrolase with broadsubstrate specificity including neutral glycerides and phospholipids.To obtain further insight into the mechanism of action of thisenzyme, we tested several sulfonyl fluorides as active site-directedinhibitors. The enzyme is resistant to the well-known serineprotease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF),but is rapidly inactivated by hexadecylsulfonyl fluoride. Thekinetics of inactivation were studied in Triton X-100 micelles.Inactivation is fast and the rate of inactivation is constantover the pH range where this lipase is active. Metal ions likeCa²⁺ and Sr²⁺ do not appreciably influence the rate of inactivation,although the enzymatic activity is significantly increased,suggesting a structural role for these ions. The S.hyicus lipasecontains a consensus sequence G-H/Y-S-X-G. Substitution by site-directedmutagenesis of this serine (Ser369) by a cysteine resulted ina mutant with only 0.2% residual activity. The activity of thismutant could not be inhibited with water-soluble sulfhydrylreagents either in the presence or absence of Triton X-100 micelles.In the presence of Triton X-100 micelles, inactivation of themutant occurred with 4-nitrophenylhexadecyl disulfide (t_1/2= 125 min) while the wild-type enzyme does not react at all.We conclude that Ser369 is the active site residue and thatin water this residue is inaccessible. Only after interfacialactivation Ser369 (or Cys369) becomes exposed and reacts withirreversible inhibitors.     

A theoretical study of substrate-induced activation of dienelactone hydrolase

Dienelactone hydrolase (DLH), an enzyme from the ß-ketoadipatepathway, catalyses the hydrolysis of dienelactone to maleylacetate.DLH is unusual because it is the only known naturally occurringenzyme which contains the catalytic triad Cys...His...Asp. Thistriad has previously been created artificially in the mutantserine proteases, thiol subtilisin and thiol trypsin. In bothcases the mutant enzymes exhibited activities several ordersof magnitude lower than the wild type enzymes; the low reactivityhas generally been attributed to the inability of these enzymesto form a catalytically active thiolate anion (Cys^– ...His⁺...Asp^–).The crystal structure of DLH suggests that the native enzymeexists predominantly in a catalytically inert configuration;the triad cysteine is neutral and points away from the activesite binding cleft. However, a crystallographic analysis ofC123S DLH complexed with an isostructural inhibitor (dienelactam)indicates that substrate binding induces a prototropic rearrangementof the active site prior to catalysis which results in the formationof a highly nucleophilic thiolate anion. We have performed abinitio SCF/MP2 calculations on a relatively small portion ofthe active site of DLH to examine the details of this activationprocess. Our calculations provide supporting evidence that theconformational changes observed in the crystal structure dueto inhibitor (or substrate) binding facilitate the formationof a reactive thiolate anion. In particular, substrate bindingalters the position of Glu36; the carboxylate side chain ofGlu36 is pushed towards C123 enabling it to abstract the thiolproton thus creating a catalytically active thiolate anion.The calculations also provide a possible explanation for thelow reactivities observed in the mutant serine proteases.     

含盐污水SBR法生物脱氮模糊控制参数

为考察含盐污水SBR法生物脱氮实现模糊控制的可行性和有效性,以实际含盐生活污水作为研究对象,考察了脱氮过程DO、pH变化规律以及盐度冲击下pH的变化规律。结果表明含盐污水SBR法脱氮过程DO曲线变化不规律,应采用pH值为模糊控制参数。不同盐度下pH曲线有相似规律,曲线上出现的反映生物脱氮进程特征点：跃升点a（break point）、氨氮谷点b (ammonia valley)、硝酸盐峰c（nitrate apex）与有机物降解结束、硝化完成和反硝化完成有很好的对应关系;盐度升高后a、b推后出现;10 g&#8226;L^-1系统受盐度冲击时pH曲线依然遵从变化规律,但随着冲击盐度的升高特征点出现位置有所变化。冲击盐度高于30 g&#8226;L^-1,pH曲线逐渐趋于平缓,特征点a、b不明显但延长曝气时间时能够出现, c点受影响较小。     

焦化废水中硫氰化物的生物降解及其与苯酚、氨氮的交互影响

硫氰化物（SCN^-）在焦化废水中的普遍出现,对COD、色度及NH⁺₄-N等指标构成贡献,且生物降解过程中还与其他污染物产生交互作用,影响工程工艺的选择和达标的控制。本研究采用实际工程不同单元工艺的活性污泥,在研究SCN-的基本降解特性与动力学基础上,重点考察苯酚对SCN^-降解及SCN^-对氨氮硝化过程的影响,评价SCN^-在焦化废水实际降解过程中与其他污染物的交互作用。研究发现,在特征活性污泥培养条件下,SCN^-的降解速率达20.15 mg SCN^-·（g MLSS）^-1·h^-1,污泥活性不受SCN^-底物浓度抑制,降解过程符合Michaelis-Menten动力学模型;苯酚对SCN^-的降解表现为毒性抑制且存在浓度阈值,高浓度苯酚可严重抑制SCN^-的降解,738 mg·L^-1苯酚使108 mg·L^-1SCN^-完全降解时间从1.5 h延长至20 h;SCN^-对硝化过程有抑制作用,可同时影响NH⁺₄的去除和NO^-₂的转化,导致硝化系统中NO^-₂浓度的积累。结果表明,生物过程中SCN^-与酚类、NH⁺₄之间的交互影响使焦化废水的处理变得复杂且难以控制,针对实际工程,需要明确各核心组分之间的相互作用,从共基质效应或毒性效应方面考虑污泥活性与浓度区间的适配,才能构建出各项污染指标得到优化控制的高效工艺。     

亚氨基芪的合成及氯甲酰基保护基脱除新工艺

对在基因工程,材料学等方面有应用的重要合成原料亚氨基芪的合成工艺进行了新的探索。以亚氨基二苄为原料,在甲苯中和三光气回流反应10h,得到酰氯亚氨基二苄,然后经溴化,脱溴化氢得到亚氨基芪甲酰氯,最后在异丙醇的氢氧化钠溶液中脱保护,高收率得到亚氨基芪,各步反应的最佳反应条件和收率(反应温度,反应时间,摩尔收率)分别为,酰氯化： 110℃, 10 h, 93.0%; 溴化、脱溴化氢：100℃, 2h而后130℃, 5h, 88.0%; 脱保护： 40℃, 3h, 98.2%,三步总收率达到80.4%。标题化合物通过¹H NMR确证结构,同时研究了酰氯作为保护基团脱去的较佳工艺条件和酰氯作为保护基保护胺基的应用。     

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild- type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.