Truong Ngoc Thanh, Naseri Joseph Itor, Vogel Andreas, Rompel Annette, Krebs B
Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany.
Arch Biochem Biophys. 2005 Aug 1;440(1):38-45. doi: 10.1016/j.abb.2005.05.024.
Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.
紫色酸性磷酸酶是双核金属酶，可催化正磷酸单酯转化为醇和正磷酸盐。来自红芸豆的这种酶的特征是具有一个Fe(III)-Zn(II)活性中心。到目前为止，针对紫色酸性磷酸酶假定的反应机制认为，位于双金属活性位点附近的两个氨基酸至关重要。根据芸豆紫色酸性磷酸酶（kbPAP）的氨基酸序列，残基H296和H202被认为对该酶的催化功能至关重要。在本研究中，已阐明了残基H202的作用。通过定点诱变制备了突变体H202A和H202R，并在杆状病毒感染的昆虫细胞中表达。基于动力学研究，假定残基H202在稳定过渡态中发挥作用，特别是在电荷补偿、底物的空间定位以及通过使底物离去基团质子化促进产物释放方面。该研究证实了H202的重要性，并阐明了其在kbPAP催化机制中的功能作用。
