Nitesh V Sule1, Angel Ugrinov2, Sanku Mallik3, D K Srivastava4. 1. Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States. Electronic address: nitesh@tamu.edu. 2. Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States. 3. Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58108, United States. 4. Department of Chemistry & Biochemistry, North Dakota State University, Fargo, ND 58108, United States. Electronic address: dk.srivastava@ndsu.edu.
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. GENERAL SIGNIFICANCE: 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.
BACKGROUND:Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the Escherichia colimethionineaminopeptidase (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. GENERAL SIGNIFICANCE: 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: Sylvie G Bernier; Nazbeh Taghizadeh; Charles D Thompson; William F Westlin; Gerhard Hannig Journal: J Cell Biochem Date: 2005-08-15 Impact factor: 4.429
Authors: Xiaochun Chen; Curtis R Chong; Lirong Shi; Tadashi Yoshimoto; David J Sullivan; Jun O Liu Journal: Proc Natl Acad Sci U S A Date: 2006-09-18 Impact factor: 11.205
Authors: Pent Zhang; Diarmuid E Nicholson; Janusz M Bujnicki; Xinzhuan Su; James J Brendle; Michael Ferdig; Dennis E Kyle; Wilbur K Milhous; Peter K Chiang Journal: J Biomed Sci Date: 2002 Jan-Feb Impact factor: 8.410
Authors: Qi-Zhuang Ye; Sheng-Xue Xie; Ze-Qiang Ma; Min Huang; Robert P Hanzlik Journal: Proc Natl Acad Sci U S A Date: 2006-06-12 Impact factor: 11.205
Authors: S M Arfin; R L Kendall; L Hall; L H Weaver; A E Stewart; B W Matthews; R A Bradshaw Journal: Proc Natl Acad Sci U S A Date: 1995-08-15 Impact factor: 11.205
Authors: P L Irwin; P E Pfeffer; L W Doner; G M Sapers; J D Brewster; G Nagahashi; K B Hicks Journal: Carbohydr Res Date: 1994-03-18 Impact factor: 2.104