Vicario P P, Green B G, Katzen H M
J Antibiot (Tokyo). 1987 Feb;40(2):209-16. doi: 10.7164/antibiotics.40.209.
The biosynthesis from L-alanine of D-alanyl-D-alanine, required for the peptidoglycan layer of the cell wall of many bacterial species, is catalyzed by two enzymes in series, alanine racemase and D-alanine: D-alanine ligase. A simple in vitro method, called the combined assay, for simultaneously testing for effectors of either or both enzymes in a single assay by coupling these enzymes to each other is described here. The experiments used to derive the optimum conditions for the assay are also described. Each enzyme is included in the assay in rate-limiting amounts, wherein the product of the initial racemase reaction, D-alanine, becomes the substrate for the subsequent ligase. The product of the overall reaction, [14C]-D-alanyl-D-alanine, is separated chromatographically from the L-[1-14C]alanine substrate, and from any D-[1-14C]alanine intermediate, at the end of the incubation, is counted and the percent conversion of substrate to product calculated. The inhibitory effects of 3-fluoro-D-alanine-2d, a known inhibitor of the racemase, and D-cycloserine and DL-1-aminoethylphosphonic acid, inhibitors of both enzymes, were readily detectable. The sensitivity of the combined assay to these inhibitors appears similar to that of earlier assays. This assay has the advantage over previous ones of being able to detect inhibitors of either enzyme in a single assay, thereby avoiding the need to screen each compound in a separate assay of each enzyme.
许多细菌细胞壁肽聚糖层所需的D -丙氨酰 - D -丙氨酸由L -丙氨酸生物合成，这一过程由两种串联的酶催化，即丙氨酸消旋酶和D -丙氨酸：D -丙氨酸连接酶。本文描述了一种简单的体外方法，称为联合测定法，通过将这些酶相互偶联，在单一测定中同时检测一种或两种酶的效应物。还描述了用于确定该测定最佳条件的实验。每种酶在测定中以限速量加入，其中初始消旋酶反应的产物D -丙氨酸成为后续连接酶的底物。在孵育结束时，通过色谱法将总反应产物[14C] - D -丙氨酰 - D -丙氨酸与L -[1 - 14C]丙氨酸底物以及任何D -[1 - 14C]丙氨酸中间体分离，计数并计算底物到产物的转化率。消旋酶的已知抑制剂3 -氟 - D -丙氨酸 - 2d以及两种酶的抑制剂D -环丝氨酸和DL - 1 -氨基乙基膦酸的抑制作用很容易检测到。联合测定法对这些抑制剂的敏感性似乎与早期测定法相似。该测定法相对于以前的方法具有优势，能够在单一测定中检测任何一种酶的抑制剂，从而避免了在每种酶的单独测定中筛选每种化合物的需要。
