Tsuchiya Tohru, Suzuki Takuo, Yamada Takafumi, Shimada Hiroshi, Masuda Tatsuru, Ohta Hiroyuki, Takamiya Ken-ichiro
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8501 Japan.
Plant Cell Physiol. 2003 Jan;44(1):96-101. doi: 10.1093/pcp/pcg011.
Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.
迄今已克隆出的叶绿素酶（Chlases）含有一个脂肪酶基序，其具有甘油三酯脂肪酶催化三联体中的活性丝氨酸残基。丝氨酸水解酶中催化丝氨酸残基的特异性抑制剂，其中包括脂肪酶，可有效抑制重组藜（Chenopodium album）叶绿素酶（CaCLH）的活性。基于此证据，我们推测叶绿素酶的水解催化机制可能与丝氨酸水解酶相似，后者在其活性位点具有由丝氨酸、组氨酸和天冬氨酸组成的催化三联体。因此，我们对假定的催化残基（Ser162）以及保守氨基酸残基（组氨酸、天冬氨酸和半胱氨酸）进行突变，以生成重组CaCLH突变体。确定了对叶绿素酶活性至关重要的三个氨基酸残基（Ser162、Asp191和His262）。这些结果表明叶绿素酶是一种丝氨酸水解酶，并且通过类比丝氨酸水解酶可能的催化机制，我们提出了叶绿素酶催化水解的机制。
