Sule, Nitesh V.; Ugrinov, Angel; Mallik, Sanku; Srivastava, D. K.
Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1850:141-149, JAN 2015

Background: Methiony1-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the Escherichia coli methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme's inhibitors, we observed that 2hydroxypropy1-beta-cyclodextrin (HP-beta-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-beta-CD. Methods: The mechanistic path for the HP-beta-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies. Results: X-ray crystallographic data of the p-cyclodextrin substrate (beta-CD-MetAMC) complex reveal that while the AMC moiety of the substrate is confined within the CD cavity, the methionine moiety protrudes outward. The steady-state kinetic data for inhibition of MetAP by HP-beta-CD-MetAMC conform to a model mechanism in which the substrate is "bridged" between HP-beta-CD and the enzyme's active-site pocket, forming HP-beta-CDMetAMC-MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-beta-CD-bound MetAMC substrate does not reach within the proximity of the enzyme's catalytic metal center, and thus the substrate fails to undergo cleavage. Conclusions: The data presented herein suggests that the bridging of the substrate between the enzyme and HP-beta-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity. General significance: Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-beta-CD mediated delivery of drugs under pathophysiological conditions. (C) 2014 Elsevier B.V. All rights reserved.

DOI:10.1016/j.bbagen.2014.10.016

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