Chang Hui-Chuan, Kung Camy C-H, Chang Tzu-Ting, Jao Shu-Chuan, Hsu Yu-Ting, Li Wen-Shan
Institute of Chemistry, Academia Sinica, Taipei, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
PLoS One. 2018 Jan 19;13(1):e0190816. doi: 10.1371/journal.pone.0190816. eCollection 2018.
Aminopeptidase P, a metalloprotease, targets Xaa-Proline peptides for cleavage [1-4]. There are two forms of human AMPP, a membrane-bound form (hmAMPP) and a soluble cytosolic form (hcAMPP)[5]. Similar to the angiotensin-I-converting enzyme, AMPP plays an important role in the catabolism of inflammatory and vasoactive peptides, known as kinins. The plasma kinin, bradykinin, was used as the substrate to conduct enzymatic activity analyses and to determine the Michaelis constant (Km) of 174 μM and the catalytic rate constant (kcat) of 10.8 s-1 for hcAMPP. Significant differences were observed in the activities of Y527F and R535A hcAMPP mutants, which displayed a 6-fold and 13.5-fold for decrease in turnover rate, respectively. Guanidine hydrochloride restored the activity of R535A hcAMPP, increasing the kcat/Km 20-fold, yet it had no impact on the activities of the wild-type or Y527F mutant hcAMPPs. Activity restoration by guanidine derivatives followed the order guanidine hydrochloride >> methyl-guanidine > amino-guanidine > N-ethyl-guanidine. Overall, the results indicate the participation of R535 in the hydrogen bond network that forms a proton relay system. The quaternary structure of hcAMPP was determined by using analytical ultracentrifugation (AUC). The results show that alanine replacement of Arg535 destabilizes the hcAMPP dimer and that guanidine hydrochloride restores the native monomer-dimer equilibrium. It is proposed that Arg535 plays an important role in hcAMMP catalysis and in stabilization of the catalytically active dimeric state.
氨肽酶P是一种金属蛋白酶，作用于Xaa-脯氨酸肽进行切割[1-4]。人氨肽酶P有两种形式，一种是膜结合形式（hmAMPP），另一种是可溶性胞质形式（hcAMPP）[5]。与血管紧张素I转换酶类似，氨肽酶P在炎症和血管活性肽（即激肽）的分解代谢中起重要作用。血浆激肽缓激肽被用作底物进行酶活性分析，确定hcAMPP的米氏常数（Km）为174μM，催化速率常数（kcat）为10.8 s-1。观察到Y527F和R535A hcAMPP突变体的活性存在显著差异，其周转速率分别降低了6倍和13.5倍。盐酸胍恢复了R535A hcAMPP的活性，使kcat/Km增加了20倍，但对野生型或Y527F突变体hcAMPP的活性没有影响。胍衍生物恢复活性的顺序为盐酸胍>>甲基胍>氨基胍>N-乙基胍。总体而言，结果表明R535参与了形成质子传递系统的氢键网络。通过分析超速离心（AUC）确定了hcAMPP的四级结构。结果表明，用丙氨酸取代Arg535会使hcAMPP二聚体不稳定，而盐酸胍可恢复天然的单体-二聚体平衡。有人提出，Arg535在hcAMMP催化和催化活性二聚体状态的稳定中起重要作用。
