Ensor C M, Tai H H
Division of Medicinal Chemistry and Pharmaceutics, University of Kentucky College of Pharmacy, Lexington 40536-0082.
Biochim Biophys Acta. 1994 Sep 21;1208(1):151-6. doi: 10.1016/0167-4838(94)90172-4.
NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. In this study the cDNA for human placental 15-PGDH was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity and characterized. The N-terminus of the recombinant protein was sequenced and found to be identical with the known amino-acid sequence of 15-PGDH. Determinations of Km and Vmax values for a number of the prostaglandins and NAD+ indicate that the recombinant enzyme does not appear to be kinetically different from the human placental enzyme. Site-directed mutagenesis was used to examine the importance of two residues which are highly conserved in the short-chain dehydrogenases which are known to be related to 15-PGDH. Tyrosine-151 was changed to phenylalanine and serine while lysine-155 was changed to glutamine and leucine. Western blot analysis indicated that the mutant and wild-type proteins were expressed at the similar levels. However, all of the mutant proteins were found to be inactive. These results indicate that both tyrosine-151 and lysine-155 are required for 15-PGDH activity.
烟酰胺腺嘌呤二核苷酸（NAD⁺）依赖性15-羟基前列腺素脱氢酶（15-PGDH）催化前列腺素分解代谢途径的第一步。该酶氧化前列腺素的15-羟基以产生通常无生物活性的15-酮代谢物。在本研究中，人胎盘15-PGDH的cDNA在大肠杆菌中表达，重组酶被纯化至同质并进行了特性鉴定。对重组蛋白的N端进行测序，发现其与15-PGDH的已知氨基酸序列相同。对多种前列腺素和NAD⁺的Km和Vmax值的测定表明，重组酶在动力学上似乎与胎盘酶没有差异。使用定点诱变来研究在已知与15-PGDH相关的短链脱氢酶中高度保守的两个残基的重要性。酪氨酸-151被替换为苯丙氨酸和丝氨酸，而赖氨酸-155被替换为谷氨酰胺和亮氨酸。蛋白质印迹分析表明，突变体和野生型蛋白以相似水平表达。然而，所有突变蛋白均无活性。这些结果表明，酪氨酸-151和赖氨酸-155都是15-PGDH活性所必需的。
