Synthetic and Conformational Studies of Active Site Peptides of Amino Acid Racemases

氨基酸消旋酶活性位点肽的合成与构象研究

• 批准号: 01571155
• 负责人: TAKEDA Yoshio
• 金额: $ 1.28万
• 依托单位: The University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01571155/
• 关键词: amino acid; racemization; active site; peptide; synthesis; conformation

D-alanine, an essential constituent of bacterial cell wall pepti-deglycan, is biosynthesized from L-Alanine by the mediation of amino acid racemase. Amog the amino acid racemase, a thermostable alanine racemase from Bacillus stearothermophilus, two alanine racemases coded by dad B and dal genes fromSalmonella typhimurium, and amino acid racemase from Pseudomonas striata were relatively well studied enzymatically. The amino acid sequences around the lysine residue to which the cofactor pyridoxal phosphate linked were elucidated as follows.B. stearothermophilus : Ala-ValーVal-Lys-Asn-Asn-Ala-Tyr (former sequence : AlaーPro-ProーLys-Ala-Asn-Ala-Tyr) ; Salmonella typhimurium : dad B Val-Trp-Ser-Val-ValーLys-Ala-Asn-Ala-Tyr-Gly-His-Gly-Ile, dal Leu-Val-Ala-Val-Val-Lys Ala-Asn-Ala-Tyr-Gly-His-Gly-Leu ; P. striata : Leu-Thr-Ala-Val-Leu-Lys-Ala-Ala-Asp-Ala-Try-Gly-His-GlyーIle.In order to obtain the basic concept in developing new inhibitor (antibacterial agents), We started the synthetic and conformational studies of active site peptides of these enzymes as model active site. At first, we compared the amino acid sequences and divided the sequences in several fragment peptides. The synthetic fragment peptides were then condensed to give protected two kinds of octapeptides and three kinds of tetradecapeptides. All the synthetic procedures were performed in the solution phase. After the N-protective group was converted to N-acetyl group, the protective groups were then removed by treatment of trimethylsilyltriflate-thioanisole system to give acetyl derivatives of two octa-and three tetradecapeptides corresponding to the amino acid sequences of active sites of above mentioned racemases. The conformational studies using NMR spectroscopy for Boc-Leu-Thr-Ala-Val-Leu-OMe and Boc-Ala-Pro-Pro-Lys (Z) -Ala-Asn-Ala-Tyr (Bzl) -OBzl were also performed.

D-丙氨酸是细菌细胞壁肽降糖的重要组成部分，由L丙氨酸在氨基酸外消旋酶的作用下生物合成。对嗜热脂肪芽孢杆菌的耐热丙氨酸消旋酶、鼠伤寒沙门氏菌的DAD B和Dal基因编码的两种丙氨酸消旋酶以及条纹假单胞菌的氨基酸消旋酶进行了较好的酶学研究。与辅因子磷酸吡哆醛连接的赖氨酸残基周围的氨基酸序列如下：B.stearthermexilus：ALA-Val-ーVal-Lys-Asn-Asn-Ala-Tyr(原序列：Ala-ーPro-ーLys-Ala-Asn-Ala-Tyr)：鼠伤寒沙门氏菌：Dad B Tal Leu-Val-Ala-Val-Val-Lys Ala-Asn-Ala-Tyr-Gly-His-Gly-Leu；Leu-Thr-Ala-Val-Leu-Lys-Ala-Ala-Asp-Ala-Try-Gly-His-GlyーIle.In为了获得开发新的抑制剂(抗菌剂)的基本概念，我们开始了这些酶的活性部位多肽的合成和构象研究，作为模型活性部位。首先，我们比较了氨基酸序列，并将其划分为几个片段多肽。将合成的片段多肽缩合，得到两种受保护的八肽和三种十四肽。所有的合成步骤都在溶液阶段进行。在N-保护基转变为N-乙酰基后，用三甲基硅烷-三氟化硫苯甲醚体系去除保护基，得到两个八肽和三个十四肽的乙酰基衍生物，与上述外消旋酶活性部位的氨基酸序列相对应。用核磁共振波谱研究了Boc-Leu-Thr-Ala-Val-Leu-Ome和Boc-Ala-Pro-Lys(Z)-Ala-Asn-Ala-Tyr(Bzl)-OBzl的构象。