Abdus Sattar A K, Yoshimoto T, Tsuru D
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Arch Biochem Biophys. 1989 Oct;274(1):241-50. doi: 10.1016/0003-9861(89)90436-0.
An aminopeptidase (EC 3.4.11.1) was purified from the extract of Lyophyllum cinerascens by ammonium sulfate fractionation and sequential chromatographies on DEAE-Sephadex, Sephadex G-150, HPLC-phenyl-5PW, and HPLC-DEAE-5PW columns, with an activity recovery of 4.6% using Leu-beta-naphthylamide as a substrate. The enzyme was a tetrameric protein of molecular weight 150,000 and was found to be rich in histidine. It exhibited a pH optimum of 7.2 and stability between pH 5.7 and 7.7. The isoelectric point of the enzyme was 4.6. The enzyme catalyzed the hydrolysis of amino acid beta-naphthylamides, Phe greater than Leu greater than Met greater than Tyr greater than Ala greater than Glu, and the differences of the measured kcat's ranged over 2-3 orders of magnitude while many of the amino acid beta-naphthylamides were not hydrolyzed at all. Other interesting comparisons include two aliphatics, Ala vs Leu, and the aromatics, Tyr vs Phe, which show a 30-fold difference in the kcat/Km values. The enzyme also hydrolyzed Leu-Gly-Gly and the B chain of oxidized insulin to release N-terminal leucine and phenylalanine, respectively. The release of N-terminal Phe from the oxidized B chain is interesting in view of the fact that the penultimate residue is Val, an unfavorable amino acid in the beta-naphthylamide series. The enzyme seems to be a true aminopeptidase, requiring the free amino groups and hydrolyzing dipeptide and oligopeptide from the N-terminal end. The enzyme was resistant to the action of amastatin. Neither sulfhydryl reagents nor serine protease inhibitors affected the enzyme activity; however, the enzyme was inhibited weakly by EDTA and bestatin and strongly by diethyl pyrocarbonate.
通过硫酸铵分级沉淀以及在DEAE-葡聚糖凝胶、葡聚糖凝胶G-150、高效液相色谱苯基-5PW和高效液相色谱DEAE-5PW柱上的连续色谱法，从灰褐环柄菇提取物中纯化出一种氨肽酶（EC 3.4.11.1），以亮氨酸-β-萘酰胺为底物时活性回收率为4.6%。该酶是一种分子量为150,000的四聚体蛋白，富含组氨酸。其最适pH为7.2，在pH 5.7至7.7之间稳定。该酶的等电点为4.6。该酶催化氨基酸β-萘酰胺的水解，苯丙氨酸＞亮氨酸＞甲硫氨酸＞酪氨酸＞丙氨酸＞谷氨酸，测得的kcat差异范围超过2至3个数量级，而许多氨基酸β-萘酰胺根本不被水解。其他有趣的比较包括两种脂肪族氨基酸，丙氨酸与亮氨酸，以及芳香族氨基酸，酪氨酸与苯丙氨酸，它们的kcat/Km值相差30倍。该酶还能水解亮氨酸-甘氨酸-甘氨酸和氧化胰岛素的B链分别释放N端亮氨酸和苯丙氨酸。考虑到倒数第二个残基是缬氨酸，在β-萘酰胺系列中是一种不利的氨基酸，从氧化B链中释放N端苯丙氨酸很有意思。该酶似乎是一种真正的氨肽酶，需要游离氨基并从N端水解二肽和寡肽。该酶对氨肽素的作用具有抗性。巯基试剂和丝氨酸蛋白酶抑制剂均不影响该酶活性；然而，该酶受到EDTA和苯丁抑制素的弱抑制，受到焦碳酸二乙酯的强烈抑制。
