Metallo-Peptides. Syntheses and Reactivities of Motif Peptides for the Active Site of Hydrolases

金属肽。

• 批准号: 13640565
• 负责人: YAMAMURA Takeshi
• 金额: $ 1.92万
• 依托单位: Tokyo University of Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13640565/
• 关键词: Hydrolases; Active-site motif; Metallo-peptides; Hydrophobicity; Dixon plot; Proton dissociative groups; Micelles; Fluorescent probes; 活性中心モチーフ; 活性中心; コンセンサスペプチド; 亜鉛錯体; コバルト錯体; 加水分解活性; 複核構造; XAFS

It is well known that native peptidases and their mutants show bell-shaped pH dependent activity. From the simulation of log(k_<cat>/K_m)-pH plots, several reaction mechanisms were proposed, and the meaning of the pK_a values that define the plots were discussed ; however, the values were not corroborative because of the lack of effective experiments. It is difficult to obtain the pKa of Zn-OH_2 in the active site, where Zn-OH_2, substrate, and other proton-dissociative groups such as Im and -C00^-are forming complex hydrogen bonding knot. In the present study, we simplified the subject from enzyme to Zn^<2+> -containing short peptides in order to derive the pK_a of Zn-OH_2. For this purpose, we focused our attention on the motif sequence of Metzincin-clan peptidases, which has an eleven-residue sequence HE-X_2-H-X_2-G-X_2-H in the catalytic zinc-binding site.Kinetic studies on AHEITHAVGMEHP/Zn^<2+> using equimolar amount of p-nitrophenylphosphate (p-NPP) as substrate in H_2O/DMSO = 1: … More 1 indicated that the motif-peptide system obeys a step-wise mechanism represented byE+S【double arrow】^^<k+1>__<k-1> E・S →^^<k+2> E+P1+P2 →^^<k+3> E・P1 (1)This study afforded a well defined log(k_<+2>/K_m)-pH plot, from which pK_a = 8.0 and 9.3 were derived as those indicating the pH window of the hydrolase activity of this system. In order to clarify the pK_a of the imidazole NH protons, pH-dependent chemical shifts of imidazole CH(δ) were studied by NMR, and then based on the pK_a values thus obtained, potentiometry experiments were performed on MTZ13/Zn^<2+>. Potentiometry afforded pK_a= 8.0 for (H_4MTZ13)Zn-(H_20)_2 【double half arrows】 (H_4MTZ13)Zn-(H_2O)(OH) and 9.5 for (H_4MTZ13)Zn(OH)_2 【double half arrows】 (H_3MTZ13)Zn-(HO)_2. Consequently, our study showed that zinc hydroxide model is also available to our motif peptide systems.In order to increase the hydrophobicity of surrounding media under aqueous conditions, thereby to avoid precipitation and to suppress conformational fluctuation due to hydrogen-bond exchange, we applied micellar systems for MTZ13/Zn^<2+>. CD experiments using cetyltrimetylammonium chloride afforded well defined spectra suggestive of robust structures (helicity > 60 %). Preliminary fluorescence studies using motif peptides with Trp in the sequence showed that the motif peptides are buried in the hydrophobic area of micelles. Less

众所周知，天然多肽酶及其突变体表现出钟形的pH依赖性活性。通过对LOG(k&lt；cat&gt；/Km)-pH曲线的模拟，提出了几种反应机理，并讨论了定义这些曲线的pKa值的意义，但由于缺乏有效的实验，这些值并不能得到证实。在活性中心，锌氢氧化物、底物和其他质子解离基团如Im和-C00^-形成复杂的氢键结，很难得到锌氢氧化物的pKa。在本研究中，我们把研究对象从酶简化为含锌的短肽，从而推导出锌氢氧化锌的pK_a。为此，我们将注意力集中在梅津菌素家族多肽酶的基序序列上，该基序在催化锌结合部位具有11个残基序列HE-X_2-H-X_2-G-X_2-H。在H_2O/DMSO=1：…中以等摩尔量的对硝基苯磷酸为底物摩尔1表明基序-多肽体系遵循BYE+S[双箭头]^&lt；k+1&gt；__&lt；k-1&gt；E·S→^&lt；k+2&gt；E+P1+P2→^&lt；k+3&gt；E·P1(1)-pH图，pKa分别为8.0和9.3。为了阐明咪唑NH质子的pKa，用核磁共振方法研究了咪唑CH(δ)随pH变化的化学位移，并根据得到的pKa值，对MTZ13/Zn^&lt；2+&gt；电位法测得(H_4MTZ13)锌-(H_20)_2[双半箭](H_4MTZ13)锌-(H_2O)(OH)的pK_a=8.0，(H_4MTZ13)锌(OH)_2[双半箭](H_3MTZ13)锌-(Ho)_2的pK_a=9.5。因此，我们的研究表明，氢氧化锌模型也适用于我们的模体多肽系统。为了在水条件下增加周围介质的疏水性，从而避免沉淀和抑制氢键交换引起的构象波动，将胶束体系应用于MTZ13/Zn^&lt；2+&gt；使用十六烷基三甲基氯化铵的CD实验提供了明确的光谱，暗示了坚固的结构(螺旋度&GT；60%)。利用序列中含有Trp的基序多肽进行的初步荧光研究表明，基序多肽被埋在胶束的疏水区。较少

项目成果

T.Suzuki et al.: "Minimal peptide model for the active site of a Zn peptidase, Stromelysin-1 Its solubilization into micelles."Peptide Science 2003. 421-424 (2004)

T.Suzuki 等人：“Zn 肽酶活性位点的最小肽模型，Stromelysin-1 其溶解到胶束中。”肽科学 2003. 421-424 (2004)

T.Suzuki, et al.: "Minimal peptide model for the active site of a Zn peptidase, Stromelysin-1. Its solubiization into micelles"Peptide Science 2003. 421-424 (2004)

T.Suzuki 等人：“Zn 肽酶活性位点 Stromelysin-1 的最小肽模型。其溶解成胶束”Peptide Science 2003. 421-424 (2004)

S.Kobayashi, et al.: "Syntheses of new artificial zinc finger proteins containing trisbipyridine-ruthenium amino acid at the N-or C-terminus as fluorescent"Peptide Science 2003. 429-430 (2004)

S.Kobayashi等人：“在N-或C-末端含有三联吡啶-钌氨基酸作为荧光的新型人工锌指蛋白的合成”Peptide Science 2003. 429-430 (2004)

H.Ishizuka, M.Abe, K.Tamura, A.Onoda, T.Yamamura: "Zinc Peptides Having the Consensus Sequence of the Active Sites of Peptidases. Structures and Characterization"Peptide Science 2003. 433-434 (2004)

H.Ishizuka、M.Abe、K.Tamura、A.Onoda、T.Yamamura：“具有肽酶活性位点共有序列的锌肽。结构和表征”肽科学 2003. 433-434 (2004)

K.Kaneko et al.: "Metal-Binding Affinity of Novel Zinc Fingers with Cys-X-Y-Cys Sequence at N- or C-Terminus"Peptide Science 2003. 431-432 (2004)

K.Kaneko 等：“N-或 C-末端具有 Cys-X-Y-Cys 序列的新型锌指的金属结合亲和力”肽科学 2003. 431-432 (2004)