Kessler E, Yaron A
Eur J Biochem. 1976 Mar 16;63(1):271-87. doi: 10.1111/j.1432-1033.1976.tb10229.x.
An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.
从溶组织梭菌的培养滤液中分离出一种氨肽酶，并将其纯化至同质。已证明纯化制剂中不存在内肽酶活性。在经校准的柱上进行凝胶过滤表明，天然酶的表观分子量为340000。在恒定丙烯酰胺浓度和浓度梯度下，在十二烷基硫酸盐存在下对变性酶进行凝胶电泳，结果出现了单一成分，其分子量分别计算为51000和59000。根据交联和变性蛋白质种类的迁移率，单体的分子量为56000。特异性研究表明，该酶可从小肽和多肽中切割包括脯氨酸和羟脯氨酸在内的所有类型的N端氨基酸残基。N端氨基酸残基与脯氨酸之间形成的肽键不会被该酶切割。氨肽酶-P和梭菌氨肽酶的联合作用导致富含脯氨酸的九肽缓激肽完全水解。观察到带电荷残基和氨基酸酰胺的水解速率较低。对一般结构为X-Gly-Gly的五种三肽进行动力学研究，其中X代表亮氨酸、苯丙氨酸、缬氨酸、丙氨酸或脯氨酸，结果表明，随着X疏水侧链尺寸的增加，Km降低。脯氨酸和丙氨酸的Kcat值最高。在Pro-Gly、Pro-Gly-Pro、Pro-Gly-Pro-Pro系列中，最后一个肽是最佳底物，表明活性位点与至少四个氨基酸残基互补。酶活性依赖于二价阳离子的存在，用Mn2+和Co2+可达到最大激活。Mn2+和Co2+激活酶 的最佳pH分别为8.6和8.2。最佳温度为40℃。Zn2+、Cu2+和对汞苯甲酸可抑制氨肽酶，但二异丙基氟磷酸酯不能抑制。
