Model studies on protease-catalysed peptide synthesis using 9-fluorenylmethoxycarbonyl protected amino acid derivatives

Summary Fmoc-Phe-OMe,Fmoc-Ala-OMe andFmoc-Gly-OH were coupled with H-Leu-NH₂ under catalytic action of chymotrypsin, papain and thermolysin, respectively. The influence of different reaction media and several reaction parameters, such as reactants and enzyme concentrations as well as reaction time, on the peptide bond formation was investigated. Abbreviations: IUPAC-IUB rules for peptides are followed, see (1984) Eur. J. Biochem.138: 9. All amino acids except of Gly are ofL-configuration.DMF=N,N-dimethylformamide,DTE=dithioerythritol,EDTA=ethylenediaminetetraacetic acid, disodium salt,N/C=ratio nucleophile component/carboxyl component,-OMe=methyl ester,TLC=thin layer chromatography.     

Discussion of the catalytic pathway of cysteine proteases based on AM1 calculations

The deacylation reaction of the cysteine protease papain was examined by AM1 reaction-coordinate calculations. The transition-state (TS) structure was extracted from the reaction pathway as corresponding to the maximum point along this minimum-energy pathway. Consistent with experimental kinetic data revealing that deacylation is about 60 times faster for thioester (---C(O)---S---) than dithioester (---C(S)---S---) intermediates, calculated E_a values are about 10 kcal mol⁻¹ lower for the former than the latter. The calculated partial atomic charges indicate that the C=O carbon in the thioester is a good site for nucleophilic attack whereas the corresponding C=S carbon in the dithioester is a poor site. The present calculations reveal that the enzyme's oxyanion hole contributes about 9 kcal mol⁻¹ toward reducing E_a for the anionic tetrahedral intermediate and TS structure. On the other hand, the presence of Asn in the putative Asn-His-Cys catalytic triad contributes only about l kcal mol⁻¹ toward reducing their E_a value. The presence of this Asn, however, did appear to stabilize His in its protonated form (ImH⁺) over its unprotonated form (Im). Two novel mechanisms are introduced to explain the unusual effect of a remote X substituent on the deacylation kinetics of the substrate family under consideration. The first mechanism invokes a “field effect” while the second mechanism embodies the concepts of induction and homoconjugation. The unique feature of these two mechanisms is that, unlike other proposed models, they circumvent the requirement for a close N…S interaction which has stimulated controversy.     

Papainkatalysierte kinetisch kontrollierte Peptidsynthese. Verwendung von Alkylestern des Arginins als Aminokomponente

Summary The papain-catalyzed reaction of esters of Z-alanine with various arginine esters was investigated. Using H-Arg-OPr ⁱ as a nucleophile the expected dipeptide product results in high yield. Otherwise, reactions with arginine esters of primary alkohols provide products undergoing further reactions. This allows the synthesis of N-protected peptide esters containing two or more arginine residues in a one-step reaction. The influence of reaction conditions on the process was investigated.

AbkürzungenDie IUPAC/IUB-Regeln für Aminosäure- und Peptidderivate wurden befolgt; vgl. Eur. J. Biochem.53, 1 (1975). Die verwendeten Aminosäuren hatten (L)-Konfiguration.-Glo-Gly-OH=Glykolylglycinester,-OCam=Carboxamidomethylester, -OBu ⁱ=Isobutylester,-OMe=Methylester,-OPr ⁱ=Isopropylester,Z=Benzyloxycarbonyl.     

木瓜蛋白酶交联聚体的制备及性质

采用交联酶聚体(CLEAs)技术制得了木瓜蛋白酶CLEAs, 优化了制备条件. 以纯乙醇为蛋白沉淀剂, 质量分数40%戊二醛为交联剂, 于4 ℃下对酶沉淀聚体交联16 h; 所得木瓜蛋白酶CLEAs的最适pH为6.0(游离酶最适pH=7.0), 最适温度范围由游离酶的80 ℃拓宽为50～80 ℃, 热稳定性和溶液稳定性亦明显提高; 微观形貌分析证明木瓜蛋白酶CLEAs优良的催化效能及稳定性来自于CLEAs单元所具有的高比表面积及单元内部多点共价固定的结合方式.     

以N-保护消旋氨基酸为羧基组分的酶促合成肽的研究

用不同的氨基保护的消旋氨基酸或酯作为羧基组分,在α-胰凝乳蛋白酶或木瓜蛋白酶的催化下与另一氨基酸的酰胺或酯缩合形成预期的光活性肽产物。以L-构型底物计算肽的产率为55%～83%,化合物的物理学常数与用相应的N-保护-L-氨基酸或酯为羧基组分,分别用化学法或酶促法测定所得结果一致。研究结果表明:利用酶的立体专一性,在形成肽键的过程中进行立体选择可得到光学活性的产物而不需预先拆分;只要溶剂体系中含有酶所需要的微量必需水,酶促反应在有机溶剂中可以顺利地进行。     

十二烷基硫酸钠对木瓜酶酶活性和构象的影响

采用紫外、荧光和圆二色等光谱技术研究了阴离子表面活性剂十二烷基硫酸钠（SDS）对木瓜酶酶活力及其构象变化的影响.结果表明：（1）SDS对木瓜酶酶活力的影响与pH有关,pH=8.00时,SDS对木瓜酶酶活力有增强作用,pH=6.30时,除了在SDS的临界胶束浓度（cmc）时木瓜酶酶活力得到增强外,其他浓度下SDS对木瓜酶酶活力没有影响,pH=9.50时,SDS对木瓜酶酶活力产生抑制作用.（2）SDS对木瓜酶的荧光产生淬灭,0.1cmc的SDS木瓜酶荧光峰红移,而SDS浓度≥1cmc后则使之蓝移;（3）当[SDS]＜cmc时,木瓜酶的α-螺旋、β-折叠、回转等有序结构的含量减少而无定形结构含量增加,当[SDS]＞cmc后,木瓜酶的α-螺旋、β-折叠构型含量有所增加,无定形构型含量则有所减少.有序结构的含量越大,酶活力越强.     

A resonance light scattering sensor based on bioinspired molecularly imprinted polymers for selective detection of papain at trace levels

A novel resonance light scattering sensor based on the molecularly imprinted polymers (MIPs) technique was developed for specific recognition of the trace quantities of papain (Pap). In this sensor, as the specific recognition element, an excellent biocompatibility of protein-imprinted polymer without fluorescent materials was easily prepared, which based on the effective synthesis of mussel-inspired bionic polydopamine (PDA) on the surface of SiO₂ nanoparticles (SiO₂@PDA NPs). This recognition element could capture the target protein selectively, which led to the enhancement of resonance light scattering intensity with the increasing of the target protein concentration. The sensor was applied to determine Pap in the linear concentration range of 2.0–20.0 nM with a correlation coefficient r = 0.9966, and a low detection limit of 0.63 nM. The relative standard deviation for 14 nM of Pap was 1.02% (n = 7). In addition, the specificity study confirmed the resultant Pap-imprinted SiO₂@PDA NPs had a high-selectivity to Pap, and the practical analytical performance was further examined by evaluating the detection of Pap in the dietary supplement with satisfactory results, with good recoveries of 97.5–105.3%.     

Functionalized cationic (η-arene)ruthenium(II) complexes for site-specific and covalent anchoring to papain from papaya latex. Synthesis, X-ray structures and reactivity studies

The cationic (η⁶-arene)ruthenium complexes 6-9 containing a chloroacetamide or a maleimide functional group on the arene ligand were synthesized and successfully used to introduce ruthenium(II) species to the active site of the cysteine endoproteinase papain in a site-directed and covalent fashion as shown by enzymatic and ESI-MS studies.     

化学修饰木瓜蛋白酶的酶学性质研究

采用邻苯二甲酸酐(PA)对木瓜蛋白酶进行了化学修饰,通过三硝基苯磺酸法、紫外光谱、荧光光谱及FT-IR光谱对修饰效果进行了初步表征,采用动力学方法考察了pH和温度对修饰酶水解活性和稳定性的影响,并计算了一系列动力学和热力学参数.实验结果表明:PA对木瓜蛋白酶的平均氨基修饰度为43%,未对酶的活性基团-SH发生修饰,修饰酶较原酶的紫外吸收峰和最大荧光发射峰均发生蓝移,紫外吸收强度降低、荧光强度增大;PA修饰未改变木瓜蛋白酶的最适反应温度,但将其最适反应pH由7.0提高到8.5,且酶活力也提高了约20%;PA修饰有效提高了酶的催化水解效率和酶与底物的亲和力,如40℃、最适pH条件下修饰酶的转化数kcat(3.03 s-1)和亲和力kcat/Km(1.70 s.L.g-1)均较原酶的(2.28 s-1、1.15 s.L.g-1)高,修饰酶催化水解反应的活化能Ea(25.4 kJ.mol-1)较原酶的(29.3 kJ.mol-1)低;PA修饰还明显提高了酶的pH稳定性和热稳定性,半衰期t1/2延长,酶分子的热变性活化能Ea,d由77.0 kJ.mol-1提高到94.5 kJ.mol-1.可见PA化学修饰法是一种有效改善木瓜蛋白酶的催化性质和稳定性的方法.     

化学修饰木瓜蛋白酶的固定化及性质研究

在底物保护和无底物保护下,用丁二酸酐对木瓜蛋白酶进行化学修饰,以三硝基苯磺酸法测定修饰酶的平均氨基修饰度,以棉布为载体,戊二醛为交联剂,对修饰前后的木瓜蛋白酶分别进行固定化.考察了温度、pH和表面活性剂SDS对化学修饰的固定化木瓜蛋白酶活力的影响,并与固定化天然木瓜蛋白酶进行了比较.研究表明,化学修饰固定化木瓜蛋白酶的最适反应温度为80℃;最适pH为9.0;在SDS浓度为20mg/mL时酶活也仍能保持在40%左右;米氏常数为187g/L.与天然的固定化酶相比,化学修饰的固定化木瓜蛋白酶的热稳定性、耐碱性和耐洗涤性得到了显著提高.