Effects of Ph on the Activity and Structure of Choline Oxidase from Alcaligenes Species

Vol. 55 No. 3/2008, 549–557 on-line at: www.actabp.pl Regular paper Effects of pH on the activity and structure of choline oxidase from Alcaligenes species Azadeh Hekmat1, Ali Akbar Saboury1, Ali Akbar Moosavi-Movahedi1, Hedayatollah Ghourchian1 and Faizan Ahmad2 1Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 2Centre for Interdisciplinary Re- search in Basic Sciences, Jamia Millia Islamia, New Delhi, India Received: 12 May, 2008; revised: 11 August, 2008; accepted: 18 August, 2008 available on-line: 04 September, 2008 A reversible effect of pH on the ionization of amino-acid residues at the active center of choline oxidase was observed near the optimum pH (8). Inactivation of choline oxidase took place in the pH ranges 3–6 and 9–11, in which irreversible changes in the structure occur leading to the enzyme inactivation. The first order rate constants of the enzyme’s inactivation at various pH values were estimated for the irreversible changes. The Arrhenius analysis revealed no significant changes in the activation enthalpy, while an increase in the activation entropy reflected an increase in the conformational freedom. Keywords: choline oxidase, glycine-betaine, optimum pH, Nile Red INTRODUCTION the ionization state of groups involved in catalysis, groups involved in binding of substrate, groups in- Normally, enzymes are only active over a volved in binding at site(s) other than the active site limited range of pH and in most cases a definite op- (allosteric effector sites) and groups on the substrates timum pH is observed. The fall of activity on either (Kuchel & Ralston, 1988). Michaelis and Davidsohn side of the optimum may be related to the effect of suggested that the catalytic activity is usually con- pH on the stability of the enzyme, which may be ir- fined to a relatively small range of pH, therefore it reversibly destroyed on one or both sides of the op- seems likely that only one of the many ionic forms timum pH (Dixon & Webb, 1979). The occurrence of of the enzyme (or rather of the active center) is cata- irreversible destruction of the enzyme activity can be lytically active (Michaelis & Davidsohn, 1911). There tested by exposing the enzyme to a range of pH val- is some evidence that the ionization of the protein ues and then testing the activity after readjusting the groups that are remote from the active center has pH to some standard value (Dixon & Webb, 1979). little or no effect, while the ionic state of groups in To describe completely the effects of different or close to the active centre has a very large effect pH values on the enzyme catalysis is a time-con- (Kuchel & Ralston, 1988). suming task. Many of the amino-acid side chains in Bernheim first demonstrated the presence of an enzyme are ionizable, but in environments with choline oxidase in animal tissues (Bernheim & Bern- polarities different from that of the free solution, the heim, 1933), afterward a great deal of work has been pKa’s will probably be significantly altered (Dixon & carried out to study this enzyme in details (Wil- Webb, 1979). However, experimentally, it is a simple liams & Litwack, 1952). Choline oxidase was first matter to determine values of steady-state kinetic isolated from rat liver in 1938 (Mann et al., 1938). parameters of an enzyme at various pH conditions. At that time, it was publicized that the choline oxi- The possible effects of pH are due to changes of dase system was composed of choline dehydroge- Corresponding author: Ali Akbar Saboury, Institute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384, Tehran, Iran; tel.: (98 21) 6695 6984; fax: (98 21) 6640 4680; e-mail: [email protected] Abbreviations: CD, circular dichroism; ChOx, choline oxidase; GB, glycine betaine; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis. 550 A. Hekmat and others 2008 nase, cytochrome c, and cytochrome oxidase (Mann Despite the large amount of work, under- et al., 1938). In 1977, Ikuta reported the purification standing of the detailed effect of different pH val- of the enzyme from the soil bacterium Arthrobacter ues on the activity of choline oxidase is still lacking. globiformis (Ikuta et al., 1977). In addition, the en- This inspired us to study the effect of various pH zyme was purified from other microorganisms, such conditions on the activity of choline oxidase from as Cylindrocarpon didymium (Yamada et al., 1979) and Alcaligenes species. At first, we tested the activity of Alcaligenes species (Ohta-Fukuyama et al., 1980; Ohta choline oxidase at different pH values. Since study- et al., 1983). The amino-acid sequence of choline oxi- ing the effects of temperature on the enzyme may dase from A. globiformis has been reported, but such provide a deeper insight into the physical principles information about the choline oxidase from Alcali- involved in its molecular organization (Sarraf et al., genes species is not available yet. 2004), these measurements were performed at 27 Choline oxidase (ChOx, EC 1.1.3.17), a FAD- and 37oC. Subsequently, we estimated the first order containing enzyme, catalyzes the oxidation of rate constants of the enzyme’s inactivation at vari- choline to glycine betaine (N,N,N-trimethylglycine) ous pH conditions. with betaine aldehyde as an intermediate and mo- Afterward we investigated the effects of lecular oxygen as primary electron acceptor (Scheme pH on the structure of choline oxidase. We em- 1) (Gadda, 2003). The molecular mass of the enzyme ployed Nile Red as a neutral fluorescence probe was estimated to be 66 kDa for a monomer (Ohta et to monitor pH-induced changes in the surface al., 1983). The prosthetic group of the enzyme from hydrophobicity of choline oxidase. These changes Alcaligenes species was identified as 8α-[N(3)-histi- can, in principle, produce changes in the position dyl]-FAD and the partial sequence of amino acids in or orientation of the tryptophan residues, altering the flavin peptide reported as Asp-Asn-Pro-Asn-His- their exposure to solvent and leading to an alter- (FAD)-Ser-Arg (Ohta-Fukuyama et al., 1980). nation in the quantum yield (Ajloo et al., 2007). Subsequently, to study the structural properties + + + (CH ) N CH CHO (CH3)3N CH2COOH (CH3)3N CH2CH2OH 3 3 2 of the protein further (Gozdek et al., 2008) at vari- O H O O ,H O 2 2 2 2 2 H2O2 ous pH ranges, we carried out far-UV CD (circu- Choline Betaine aldehyde Be taine lar dichroism) experiments. Gel electrophoresis of Scheme 1. Reaction catalyzed by choline oxidase. choline oxidase at different pH values was also performed. It is important to mention that this is the first The investigation on ChOx is of interest to attempt to study the changes of choline oxidase (from scientists for a number of reasons. The mechanism Alcaligenes species) activity induced by low and high of the carbon–hydrogen bond cleavage in choline pH at temperatures of 27 and 37oC. The informa- by ChOx is important because of the high energetic tion obtained in this study will help to understand barrier associated with this process (Gadda, 2003). the effects of pH on the activity and conformation of This reaction also is of considerable importance choline oxidase from Alcaligenes species. for medical and biotechnological reasons, since the accumulation of glycine betaine (GB) has been observed in a number of human pathogenic bacteria MATERIALS AND METHODS and in the cytoplasm of many plants in response to hyperosmotic and temperature stresses, prevent- Materials. Choline oxidase (EC 1.1.3.17 from ing dehydration and cell death. GB also protects Alcaligenes species), choline chloride, horseradish the transcriptional and translational machinery peroxidase (HRP) and Nile Red (9-diethylami- by decreasing the melting temperature of double- no-5H-benzo phenoxazine-5-one) were supplied stranded DNA. This seems to suggest that GB be- by Sigma (St. Louis, MO, USA). Tris and 4-ami- haves like chaperonin (Graham & Wilkinson, 1992; noantipyrene were from BDH Chemicals Ltd. and Bae et al., 1993; Kaenjak et al., 1993; Culham et al., Aldrich (St. Louis, MO, USA), respectively. Potas- 1994; Deshnium et al., 1995; Deshnium et al., 1997; sium phosphate (K2HPO4 and KH2PO4) was from Alia et al., 1998; Kempf & Bremer, 1998; Peddie et Merck (Darmstat, Germany). al., 1998; Sakamoto et al., 1998, 2000; Holmstrom et Enzyme assay. The initial velocity of the al., 2000). In addition, the development of biosen- choline oxidase-catalyzed reaction was determined sors for detection of choline, choline derivatives using the procedure described by Sigma-Aldrich in and organophosphorous compounds in biological details (Okabe et al., 1977; Keesey, 1987) in a Shi- (Tavakoli et al., 2005) and environmental samples madzu model UV-3100 spectrophotometer, equipped (including air, soil and water) (Nunes & Barcelo, with a temperature control system. A stock solution 1998; Guerrieri et al., 2002) render this enzyme of of ChOx (96.8 µM) was prepared in 10 mM Tris/HCl clinical and industrial interest. buffer (pH 8.0) containing 134 mM KCl and 2 mM Vol. 55 Effects of pH on choline oxidase from Alcaligenes species 551 EDTA. ChOx was assayed in a mixture containing RESULTS AND DISCUSSION 1 ml of 100 mM Tris/HCl (pH 8.0), 5 units/mg HRP, 50 mM 4-aminoantipyrine and 150 mM choline chlo- The pH-dependent activity changes of ride as a substrate at 37oC. The reaction was started choline oxidase by the addition of 20 µl of ChOx in a temperature- controlled cuvette compartment. The enzyme reac- The effect of pH on the activity itself can tion was monitored spectrophotometrically at 500 be determined by carrying out a series of velocity nm by the production of quinonimine dye using an measurements at different pH values using a suf- absorption coefficient, ε = 12 000 M−1 cm−1 (Okabe et ficiently high substrate concentration, which elimi- al., 1977; Keesey, 1987).

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