title: One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity.
abstract: A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.
authors.affiliation: Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Takaoka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsutsumi
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Kasagi
authors.forename: Eiji
authors.initials: E
authors.lastname: Nakata
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D006835:Hydrazones / Q000737:chemistry; D008958:Models, Molecular; D010091:Oximes / Q000737:chemistry; D015202:Protein Engineering; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006835:Hydrazones; D010091:Oximes; D013449:Sulfonamides; D024402:Carbonic Anhydrase II