Laboratory Exercises Comparison of the Interaction of Cobalt Bovine Carbonic Anhydrase II with Acetazolamide and Methazolamide and the Reaction of Apoenzyme with Cobalt(II) Complexes of Acetazolamide and Methazolamide SPECTROPHOTOMETRIC STUDY Received for publication, June 5, 2002, and in revised form, August 29, 2002 Silvia Rubio‡, Consuelo Borra ´ s§, Gloria Alzuet‡, and Joaquin Borra ´ s‡¶ From the ‡Departamento de Quı´mica Inorga ´ nica, Universitat de Vale ` ncia, Vicent Andre ´ s Estelle ´ s s/n, 46100 Burjassot, Spain and the §Departamento de Fisiologı´a Animal, Universitat de Vale ` ncia, Avda. Blasco Iban ˜ ez 15, 46010 Valencia, Spain The metalloenzyme carbonic anhydrase (CA) is an attractive choice for a research-based bioinorganic laboratory course. In this project the interaction of cobalt bovine carbonic anhydrase II (CoBCAII) with acetazolamide and methazolamide and the reaction of apoenzyme with cobalt(II) complexes of acetazol- amide and methazolamide is studied by UV-visible spectroscopy. Prior to this spectroscopic study students are given native BCAII, and they prepare apoBCAII and CoBCAII. A major aim is to provide experience in handling metalloproteins and in the study of metal complexes-protein interactions. Keywords: Carbonic anhydrase, sulfonamide inhibitors, sulfonamide-CoCA interaction, cobalt(II) complexes- apoCA interaction, UV-visible spectroscopic study. The ubiquitous enzyme carbonic anhydrase (CA) 1 is present in Archaea, prokaryotes, and eukaryotes. The comparison of sequences and crystal structures of the mammalian and plant enzymes demonstrates that they evolved independently, and the mammalian and plant en- zymes have been designated the - and -class, respec- tively. An additional independently evolved -class was reported in 1994 for which phylogenetic analyses predict an ancient origin [1]. In higher vertebrates, including hu- mans, 14 different CA isozymes have been described up to now. They are involved in the crucial physiological pro- cesses connected with respiration and transport of CO 2 / bicarbonate, pH homeostasis, electrolyte secretion, and biosynthetic reactions such as lipogenesis, gluconeogen- esis, and ureagenesis among others. In addition to the physiological reaction, the reversible hydration of carbon dioxide to bicarbonate, CAs also catalyze a variety of reactions such as aldehyde hydration and hydrolysis of carboxylic acid esters [2]. Human carbonic anhydrase II (HCAII) is a zinc enzyme predominantly found in red blood cells where it catalyzes the reaction [3, 4]: CO 2 + H 2 O N HCO 3 - + H + With a turnover rate of 10 6 /s at pH 9, 25 °C, it is one of the fastest enzymes known. The structure of HCAII has been refined at 2.0-Å resolution [5]. The active site cavity of HCAII has a conical shape. It is about 15 Å wide in entrance and penetrates about 15 Å into the middle of the molecule. The zinc ion, at the bottom of the cavity, is ligated to three histidyl residues (His-94, His-96, and His-114), and the fourth ligand is a water molecule (water 263). The zinc coordination is almost tetrahedral (see Fig. 1). CA has been widely investi- gated spectroscopically (using Co(II)-substituted enzyme), kinetically, and by x-ray crystallography. The research on CA includes the use of inhibitors of this enzyme. Sulfonamides represent an important class of biologi- cally active compounds with at least five different classes of pharmacological agents. The sulfonamides are well known inhibitors of zinc carbonic anhydrase enzyme and have many applications including use as diuretics, anti- glaucoma agents, and anti-epileptic drugs among others [6]. Among the large number of sulfonamides, acetazol- amide (H 2 acm) and its derivative, methazolamide, (Hmacm) (Scheme 1) are used extensively. ¶ To whom correspondence should be addressed. Tel.: 0034963864530; Fax: 0034963864960; E-mail: Joaquin.Borras@ uv.es. 1 The abbreviations used are: CA, carbonic anhydrase; CoB- CAII, cobalt bovine carbonic anhydrase II; HCAII, human carbonic anhydrase II; H 2 acm, acetazolamide; Hmacm, methazolamide; Hacm - , monodeprotonated form of H 2 acm; acm 2- , dideproto- nated form of H 2 acm. SCHEME 1 © 2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 31, No. 1, pp. 28 –33, 2003 This paper is available on line at http://www.bambed.org 28