title: Effects of cryoprotectants on the structure and thermostability of the human carbonic anhydrase II-acetazolamide complex.
abstract: Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277-298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20-25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.
authors: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, 1600 SW Archer Road, PO Box 100245, Gainesville, FL 32610, USA.
authors: Mayank
authors: M
authors: Aggarwal
authors: Christopher D
authors: CD
authors: Boone
authors: Bhargav
authors: B
authors: Kondeti
authors: Chingkuang
authors: C
authors: Tu
authors: David N
authors: DN
authors: Silverman
authors: Robert
authors: R
authors: McKenna
mesh_terms: D000086:Acetazolamide / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry*; D020134:Catalytic Domain; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000187:drug effects*; D005990:Glycerol / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013395:Sucrose / Q000737:chemistry; D013696:Temperature
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D003451:Cryoprotective Agents; D013395:Sucrose; D024402:Carbonic Anhydrase II; D000086:Acetazolamide; D005990:Glycerol
keywords: acetazolamide; carbonic anhydrase; cryoprotectants; glycerol; sucrose
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