title: Simultaneous retention of thermostability and specific activity in chimeric human alkaline phosphatases.
abstract: Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75°C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan, yoshiji@fides.dti.ne.jp.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Sasajima
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kohama
authors.forename: Miki
authors.initials: M
authors.lastname: Kojima-Misaizu
authors.forename: Naoya
authors.initials: N
authors.lastname: Kurokawa
authors.forename: Yuko
authors.initials: Y
authors.lastname: Hara
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.forename: Masaki
authors.initials: M
authors.lastname: Ihara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D058851:GPI-Linked Proteins / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental