Kitazono A, Kabashima T, Huang H S, Ito K, Yoshimoto T
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Arch Biochem Biophys. 1996 Dec 1;336(1):35-41. doi: 10.1006/abbi.1996.0529.
In spite of the numerous studies regarding prolyl aminopeptidase, little is known about its mechanism and the significance of its similarity to a number of hydrolases of diverse specificity that belong to the alpha/beta hydrolase-fold family (Pseudomonas 2-hydroxymuconic semialdehyde hydrolase, atropinesterase, and 2-hydroxy-6-oxophenylhexa-2,4-dienoic acid hydrolase; human and rat epoxide hydrolases). We report the cloning and sequencing of the novel prolyl aminopeptidase gene from Flavobacterium meningosepticum (FPAP) which allowed a more comprehensive sequence comparison. FPAP was found to be a 35-kDa monomeric enzyme, releasing N-terminal proline but not hydroxyproline residues from small peptides and naphthylamide esters. Using the unweighted pair group method with arithmetic mean method, an evolutionary tree that depicts the probable relationship between the prolyl aminopeptidases and the alpha/beta hydrolase-fold enzymes was constructed. Since the alpha/beta hydrolase-fold family might also include the members of the prolyl oligopeptidase family (prolyl oligopeptidase, dipeptidyl peptidase IV, and prolyl carboxypeptidase), this proposal links all the known Pro-Y bond-cleaving proline-specific peptidases (prolyl oligopeptidase family, prolyl aminopeptidases, and prolinase) as enzymes with similar scaffolds and hydrolytic mechanisms. On the other hand, the enzymes that cleave X-Pro bonds are metalloenzymes grouped within the "pita-bread" fold family (aminopeptidase P and prolidase). Although the latter two enzymes show significant sequence homology, prolyl aminopeptidase, prolinase, and the members of the prolyl oligopeptidase family do not, and might share the alpha/beta hydrolase-fold scaffold. This rationale would explain the failure in finding a common "proline-recognizing motif" in the primary structures of these proline-specific peptidases.
尽管有众多关于脯氨酰氨肽酶的研究，但对于其作用机制以及它与多种具有不同特异性的水解酶（属于α/β水解酶折叠家族，如假单胞菌2-羟基粘康酸半醛水解酶、阿托品酯酶和2-羟基-6-氧代苯并六-2,4-二烯酸水解酶；人和大鼠环氧化物水解酶）相似性的意义却知之甚少。我们报道了从脑膜炎败血黄杆菌中克隆和测序新型脯氨酰氨肽酶基因（FPAP），这使得更全面的序列比较成为可能。发现FPAP是一种35 kDa的单体酶，可从小肽和萘酰胺酯中释放N端脯氨酸，但不释放羟脯氨酸残基。使用算术平均法的非加权配对组方法构建了一个进化树，描绘了脯氨酰氨肽酶与α/β水解酶折叠酶之间可能的关系。由于α/β水解酶折叠家族可能还包括脯氨酰寡肽酶家族的成员（脯氨酰寡肽酶、二肽基肽酶IV和脯氨酰羧肽酶），这一推测将所有已知的切割Pro-Y键的脯氨酸特异性肽酶（脯氨酰寡肽酶家族、脯氨酰氨肽酶和脯氨酸酶）联系起来，认为它们是具有相似支架和水解机制的酶。另一方面，切割X-Pro键的酶是金属酶，归属于“皮塔饼”折叠家族（氨肽酶P和脯氨酰二肽酶）。尽管后两种酶显示出显著的序列同源性，但脯氨酰氨肽酶、脯氨酸酶和脯氨酰寡肽酶家族的成员却没有，它们可能共享α/β水解酶折叠支架。这一理论可以解释为何在这些脯氨酸特异性肽酶的一级结构中未能找到共同的“脯氨酸识别基序”。
