title: Bridging of a substrate between cyclodextrin and an enzyme's active site pocket triggers a unique mode of inhibition.
abstract: BACKGROUND
  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 sequestration of the substrate by HP-β-CD.
  
  
  METHODS
  The mechanistic path for the HP-β-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 β-cyclodextrin-substrate (β-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-β-CD-MetAMC conform to a model mechanism in which the substrate is "bridged" between HP-β-CD and the enzyme's active-site pocket, forming HP-β-CD-MetAMC-MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-β-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-β-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity.
  
  
  CONCLUSIONS
  Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-β-CD mediated delivery of drugs under pathophysiological conditions.
authors.affiliation: Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States. Electronic address: nitesh@tamu.edu.
authors.forename: Nitesh V
authors.initials: NV
authors.lastname: Sule
authors.affiliation: Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States.
authors.forename: Angel
authors.initials: A
authors.lastname: Ugrinov
authors.affiliation: Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58108, United States.
authors.forename: Sanku
authors.initials: S
authors.lastname: Mallik
authors.affiliation: Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States. Electronic address: dk.srivastava@ndsu.edu.
authors.forename: D K
authors.initials: DK
authors.lastname: Srivastava
mesh_terms: D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013379:Substrate Specificity; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: D003374:Coumarins; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D047392:beta-Cyclodextrins; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000626:Aminopeptidases; C028743:7-amino-4-methylcoumarin
keywords: Cyclodextrin; Cyclodextrin–substrate complex; Inhibition; Methionine aminopeptidase; Molecular modeling; Substrate bridging