Vol. 55 No. 3/2008, 549–557

on-line at: www.actabp.pl Regular paper

Effects of pH on the activity and structure of 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 ofcholine 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 en- zyme inactivation. The first order rate constants of the ’s inactivation at various pHval- ues 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 - in crease in the conformational freedom.

Keywords: choline oxidase, -betaine, optimum pH, nile red

INTRODUCTION the ionization state of groups involved in , groups involved in binding of , groups in- Normally, are only active over a volved in binding at site(s) other than the 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 groupsin To describe completely the effects of different or close to the active centre has a very large effect pH values on the 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)