Kwon H J, Amada K, Haruki M, Morikawa M, Kanaya S
Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
FEBS Lett. 2000 Oct 20;483(2-3):139-42. doi: 10.1016/s0014-5793(00)02103-7.
A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.
来自假单胞菌属MIS38（PML）的一种脂肪酶是脂肪酶家族I.3的成员。我们通过定点诱变分析了五个组氨酸残基（His(30)、His(274)、His(291)、His(313)和His(365)）以及五个酸性氨基酸残基（Glu(253)、Asp(255)、Asp(262)、Asp(275)和Asp(290)）的作用，这些残基在I.3家族脂肪酶的氨基酸序列中完全保守。我们发现，将His(313)或Asp(255)突变为丙氨酸几乎使该酶完全失活，而将其他残基突变为丙氨酸并未严重影响酶活性。远紫外和近紫外圆二色光谱的测量表明，His(313)或Asp(255)突变导致的失活并非由于三级结构的显著变化。我们提出，His(313)和Asp(255)与Ser(207)一起在PML中形成一个催化三联体。
