Premium
Rationally designed mutations of E. coli alkaline phosphatase confer selective purine derivative binding
Author(s) -
Malecha Mitchell Richard,
Weaver Todd Michael,
Grilley Daniel Patrick
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.528.5
Subject(s) - alkaline phosphatase , phosphatase , chemistry , uncompetitive inhibitor , biochemistry , purine , isozyme , non competitive inhibition , enzyme kinetics , nucleotide , stereochemistry , binding site , active site , enzyme , gene
Small organic molecules, like phenylalanine and theophylline, are effective inhibitors of mammalian alkaline phosphatases, such as calf intestinal alkaline phosphatase (CIAP). However, organic compounds do not hamper E. coli alkaline phosphatase (EcAP) activity. Sequence and structural analysis of alkaline phosphatase isozymes revealed a lack of conservation at EcAP residues that may be important for organic inhibition, thereby providing a potential explanation for the contrary inhibition. By mutating these EcAP residues to mimic analogous residues in mammalian APases, uncompetitive inhibition of EcAP by a class of aromatic molecules was conferred. While variants with single mutations are unaffected by organic effectors, variants expressing multiple mutations are inhibited, suggesting a synergistic relationship essential for organic binding. Circular dichroism was utilized to verify similar stability and kinetics for each variant. Michaelis‐Menten experiments were used to identify inhibition and confirm similar pH and cofactor dependence. The importance of key residues was determined based on their ability to confer organic inhibition. Furthermore, utilization of a set of purine derivatives allowed determination of the structure‐activity relationship for inhibitor binding. Specifically, this analysis enabled identification of hydrogen bonding requirements for organic inhibition. In total, designed substitutions altering EcAP residues near the active site to mimic analogous residues in mammalian APases confers uncompetitive inhibition by specific derivatives of a class of aromatic organic molecules. Support or Funding Information Research generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity grant (MRM). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .