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Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

ABSTRACT: Methionyl-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 2-hydroxypropyl-?-cyclodextrin (HP-?-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-?-CD.The mechanistic path for the HP-?-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.X-ray crystallographic data of the ?-cyclodextrin-substrate (?-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-?-CD-MetAMC conform to a model mechanism in which the substrate is "bridged" between HP-?-CD and the enzyme's active-site pocket, forming HP-?-CD-MetAMC-MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-?-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.The data presented herein suggests that the bridging of the substrate between the enzyme and HP-?-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity.Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-?-CD mediated delivery of drugs under pathophysiological conditions.

SUBMITTER: Sule NV

PROVIDER: S-EPMC4281286 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

Sule Nitesh V NV Ugrinov Angel A Mallik Sanku S Srivastava D K DK

Biochimica et biophysica acta 20141024 1

<h4>Background</h4>Methionyl-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 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequ ...[more]

PMID: 25450177

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Project description:(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio- and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogues designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analogue compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogues lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.

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Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

Publications

Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.

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