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Biophysical and kinetic analysis of wild‐type and site‐directed mutants of the isolated and native dehydroquinate synthase domain of the AROM protein
Author(s) -
Park Alison,
Lamb Heather K.,
Nichols Charlie,
Moore Jonathan D.,
Brown Katherine A.,
Cooper Alan,
Charles Ian G.,
Stammers David K.,
Hawkins Alastair R.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04705404
Subject(s) - chemistry , shikimate pathway , stereochemistry , isothermal titration calorimetry , active site , circular dichroism , enzyme kinetics , atp synthase , binding site , cofactor , biochemistry , enzyme
Abstract Dehydroquinate synthase (DHQS) is the N‐terminal domain of the pentafunctional AROM protein that catalyses steps 2 to 7 in the shikimate pathway in microbial eukaryotes. DHQS converts 3‐deoxy‐D‐ arabino ‐heptulosonate‐7‐phosphate (DAHP) to dehydroquinate in a reaction that includes alcohol oxidation, phosphate β‐elimination, carbonyl reduction, ring opening, and intramolecular aldol condensation. Kinetic analysis of the isolated DHQS domains with the AROM protein showed that for the substrate DAHP the difference in K m is less than a factor of 3, that the turnover numbers differed by 24%, and that the K m for NAD + differs by a factor of 3. Isothermal titration calorimetry revealed that a second (inhibitory) site for divalent metal binding has an approximately 4000‐fold increase in K D compared to the catalytic binding site. Inhibitor studies have suggested the enzyme could act as a simple oxidoreductase with several of the reactions occurring spontaneously, whereas structural studies have implied that DHQS participates in all steps of the reaction. Analysis of site‐directed mutants experimentally test and support this latter hypothesis. Differential scanning calorimetry, circular dichroism spectroscopy, and molecular exclusion chromatography demonstrate that the mutant DHQS retain their secondary and quaternary structures and their ligand binding capacity. R130K has a 135‐fold reduction in specific activity with DAHP and a greater than 1100‐fold decrease in the k cat / K m ratio, whereas R130A is inactive.