EFFECT OF CALCIUM, OTHER IONS, AND PH ON THE REACTIONS OF BARLEY PEROXIDASE WITH HYDROGEN-PEROXIDE AND FLUORIDE - CONTROL OF ACTIVITY THROUGH CONFORMATIONAL CHANGE

Transient-state kinetic analysis of compound I formation for barley gr ain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological func tion. The enzyme shows very little reaction with H2O2 at pH > 5 and ex hibited saturation kinetics at higher H2O2 concentrations (k(cat)(app) increases from 1.1 s(-1) at pH 4.5 to 4.5 s(-1) at pH 3.1 with an enz yme-linked pK(a) < 3.7 (Rasmussen, C. B., Bakovic, M., Welinder, K. G. , and Dunford, H. B. (1993) FEES Lett. 321, 102-105)). In the present paper, it is shown that the presence of Ca2+ gives rise to biphasic ki netics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typic al of a classical peroxidase (k(1)(app) = 1.5 x 10(7) M-1 s(-1), which is pH-independent between 3.3 and 5.0). The amount of enzyme reacting in the fast phase increases with Ca2+ concentration (K-d = 4 +/- 1 mM at pH 4.0), although it is also moderately inhibited by Cl-. The abso rption spectrum of BP-1, which appears to be a five-coordinate high sp in ferric in the resting state changes insignificantly in the presence of Ca2+. In the presence of Cl-, it becomes six-coordinate high spin (K-d approximately 60 mM at pH 4.0) but only if Ca2+ is also present. Fluoride binds to BP 1 with monophasic kinetics in the presence of 0-5 mM Ca2+. The activating effect of Ca2+ can be mimicked only by replac ing it with Sr2+ and Ba2+ ions. Comparing these data with the crystal structure of the inactive neutral form of BP 1 (Henriksen, k, Welinder , K. G., and Gajhede, M. (1997) J. Biol. Chem. 273, 2241-2248) and sim ilar data for wild-type and mutant peroxidases of plant and fungal ori gin suggests (i) a proton-induced conformational change from an inacti ve BP 1 at neutral pH to a low activity BP 1 form with a functional di stal histidine and (ii) a Ca2+-induced slow conformational change (at least compared with compound I formation) of this low activity form to a high activity BP 1 with a typical peroxidase reactivity. BP 1 is th e first example of a plant peroxidase whose activity can be reversibly controlled at the enzyme level by pH-and Ca2+-induced conformational changes.