Inoue Takahiko, Ito Kiyoshi, Tozaka Tomohiro, Hatakeyama Susumi, Tanaka Nobutada, Nakamura Kazuo T, Yoshimoto Tadashi
Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
Arch Biochem Biophys. 2003 Aug 15;416(2):147-54. doi: 10.1016/s0003-9861(03)00293-5.
Prolyl aminopeptidase from Serratia marcescens hydrolyzed x-beta-naphthylamides (x=prolyl, alanyl, sarcosinyl, L-alpha-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2-tert-butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K(i) value of Pro-TBODA, Ala-TBODA, and Sar-TBODA was 0.5 microM, 1.6 microM, and 12mM, respectively. The crystal structure of enzyme-Pro-TBODA complex was determined. Pro-TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.
粘质沙雷氏菌的脯氨酰氨肽酶可水解X-β-萘酰胺（X = 脯氨酰、丙氨酰、肌氨酰、L-α-氨基丁酰和正缬氨酰），这表明该酶有一个容纳五元环的口袋。基于底物特异性，合成了具有2-叔丁基-[1,3,4]恶二唑（TBODA）的脯氨酸、丙氨酸和肌氨酸的新型抑制剂。脯氨酸-TBODA、丙氨酸-TBODA和肌氨酸-TBODA的抑制常数（Ki）值分别为0.5微摩尔、1.6微摩尔和12毫摩尔。测定了酶-脯氨酸-TBODA复合物的晶体结构。脯氨酸-TBODA位于活性位点。在酶与脯氨酸抑制剂的氨基之间发现了四种静电相互作用（Glu204:0E1-N:抑制剂、Glu204:0E2-N:抑制剂、Glu232:0E1-N:抑制剂和Gly46:O-N:抑制剂），并且抑制剂的残基插入到由Phe139、Leu141、Leu146、Tyr149、Tyr150和Phe236组成的疏水口袋中。通过定点诱变证实了疏水口袋中的Phe139、Tyr149和Phe236以及静电相互作用中的Glu204和Glu232的作用，这表明脯氨酸的分子识别是通过四种静电相互作用以及在酶的疏水口袋中的插入来实现的。
