International Journal of Biological Macromolecules 27 (2000) 29–33 Enthalpy and enzyme activity of modified histidine residues of adenosine deaminase and diethyl pyrocarbonate complexes G. Ataie a , A.A. Moosavi-Movahedi a, *, A.A. Saboury a , G.H. Hakimelahi b , J.Ru. Hwu b , S.C. Tsay b a Institute of Biochemistry and Biophysics, Uniersity of Tehran, Tehran, Iran b Institute of Chemistry, Academia Sinica Taipei, Taipei, Taiwan, ROC Received 15 March 1999; accepted 14 September 1999 Abstract Kinetic and thermodynamic studies have been made on the effect of diethyl pyrocarbonate as a histidine modifier on the active site of adenosine deaminase in 50 mM sodium phosphate buffer pH 6.8, at 27°C using UV spectrophotometry and isothermal titration calorimetry (ITC). Inactivation of adenosine deaminase by diethyl pyrocarbonate is correlated with modification of histidyl residues. The number of modified histidine residues complexed to active site of adenosine deaminase are equivalent to 4. The number and energy of histidine binding sets are determined by enthalpy curve, which represents triple stages. These stages are composed of 3,1 and 1 sites of histidyl modified residues at diethyl pyrocarbonate concentrations, 0.63, 1.8, 3.3 mM. The heat contents corresponding to the first, second and third sets are found to be 18 000, 22 000 and 21 900 kJ mol -1 respectively. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Adenosine deaminase; Diethyl pyrocarbonate; Histidine residues; Chemical modification; Enthalpy www.elsevier.com/locate/ijbiomac 1. Introduction Adenosine deaminase (ADA, EC 3.5.4.4), a key en- zyme in purine metabolism, catalyzes the irreversible hydrolytic deamination of active adenosine to form the inactive metabolite inosine. It is present in virtually all mammalian cells and has a central role in maintaining immune competence of ADA, that is widely distributed in the brain [1], and is present also in neurons [2]. Lack of this enzyme is associated with severe combined im- munodeficiency disease (SCID) [3], which makes ADA a paradigm for structure — function studies of a genetic disease. ADA is also specifically involved in a variety of other diseases including acquired im- munodeficiency syndrome (AIDS), anemia, various lymphomas and leukemia [4]. ADA is a glycoprotein consisting of a single polypep- tide chain of 311 amino acids [5]. It was sequenced in 1984 [6]. The primary amino acid sequence of ADA is highly conserved across species [7]. The studies on crystal structure of mouse ADA shows the protein to be composed of an eight-stranded / motif with five additional -helices, and the active site located at the -barrel COOH terminal end [8]. The crystal structure has also revealed that ADA is a metalloenzyme that complexes one mole of Zn 2 + per mole of protein. The zinc ion, which lies in the deepest part of the active site pocket, is coordinated by five atoms, three N 2 atoms of His 15, His 17 and His 214, the O 2 of Asp 294 and the N-6 or O-6 of substrate or inhibitors. The three N 2 atoms exhibit tetrahedral geometry with O 2 and O-6 or N-6 [8]. His 17 further participated in hydrogen bonding of the ribose. Experiments confirm that in the active enzyme, zinc plays a critical role in catalysis [9]. His 238, a conserved amino acid located in hydrogen- bonding distance from C-6 of the substrate in the active site of ADA, is suggested to play an important role in catalysis by electrostatically stabilizing the hydroxylate Abbreiations: ADA, adenosine deaminase; DEP, diethyl pyrocar- bonate; TAM, thermal activity monitor. * Corresponding author. Tel.: +98-21-640-9517; fax: +98-21-640- 4680. E-mail address: moosavi@ibb.ut.ac.ir (A.A. Moosavi-Movahedi) 0141-8130/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0141-8130(99)00113-0