Ghosh M, Grunden A M, Dunn D M, Weiss R, Adams M W
Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
J Bacteriol. 1998 Sep;180(18):4781-9. doi: 10.1128/JB.180.18.4781-4789.1998.
Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.
脯氨酸二肽酶（脯氨酰二肽酶）通过多步色谱法从嗜热嗜压古菌激烈火球菌的细胞提取物中纯化得到。该酶是一种同型二聚体（每个亚基39.4 kDa），纯化后的酶每个亚基含有一个钴原子。其催化活性还需要添加Co2+离子（Kd，0.24 mM），这表明该酶有第二个金属离子结合位点。Co2+可以被Mn2+取代（导致活性降低25%），但不能被Mg2+、Ca2+、Fe2+、Zn2+、Cu2+或Ni2+取代。脯氨酰二肽酶表现出狭窄的底物特异性，仅水解C端为脯氨酸且N端为非极性氨基酸（甲硫氨酸、亮氨酸、缬氨酸、苯丙氨酸或丙氨酸）的二肽。以甲硫氨酸 - 脯氨酸为底物时，脯氨酰二肽酶的最佳活性出现在pH 7.0和温度100℃下。纯化后的脯氨酰二肽酶的N端氨基酸序列用于在激烈火球菌基因组数据库中鉴定一个推定的脯氨酰二肽酶编码基因，其产物对应349个氨基酸。该基因在大肠杆菌中表达，并纯化了重组蛋白。其性质，包括分子量、金属离子依赖性、最适pH和温度、底物特异性和热稳定性，与激烈火球菌的天然脯氨酰二肽酶无法区分。此外，天然和重组形式的底物甲硫氨酸 - 脯氨酸的Km值相当，尽管重组酶的Vmax值比天然蛋白高两倍。激烈火球菌脯氨酰二肽酶的氨基酸序列与嗜温生物的脯氨酰二肽酶有显著相似性，但该酶在底物特异性、热稳定性、金属依赖性和对抑制剂的反应方面与它们不同。激烈火球菌的这种酶似乎是双核N端外肽酶家族中第二个含钴成员（仅次于甲硫氨酸氨肽酶）。
