Abstract
Transient-state kinetic analysis of compound I formation for barley grain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological function. The enzyme shows very little reaction with H2O2 at pH > 5 and exhibited saturation kinetics at higher H2O2 concentrations (kcatapp increases from 1.1 s-1 at pH 4.5 to 4.5 s-1 at pH 3.1 with an enzyme-linked pKa < 3.7 (Rasmussen, C.B., Bakovic, M., Welinder, K. G., and Dunford, H. B. (1993) FEBS Lett. 321, 102-105)). In the present paper, it is shown that the presence of Ca2+ gives rise to biphasic kinetics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typical of a classical peroxidase (K1app = 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 (Kd = 4 +/- 1 mM at pH 4.0), although it is also moderately inhibited by Cl-. The absorption spectrum of BP 1, which appears to be a five-coordinate high spin ferric in the resting state changes insignificantly in the presence of Ca2+. In the presence of Cl-, it becomes six-coordinate high spin (Kd 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 replacing it with Sr2+ and Ba2+ ions. Comparing these data with the crystal structure of the inactive neutral form of BP 1 (Henriksen, A., Welinder, K. G., and Gajhede, M. (1997) J. Biol. Chem. 273, 2241-2248) and similar data for wild-type and mutant peroxidases of plant and fungal origin suggests (i) a proton-induced conformational change from an inactive BP 1 at neutral pH to a low activity BP 1 form with a functional distal histidine and (ii) a Ca(2+)-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 the first example of a plant peroxidase whose activity can be reversibly controlled at the enzyme level by pH- and Ca(2+)-induced conformational changes.
Highlights
Transient-state kinetic analysis of compound I formation for barley grain peroxidase (BP 1) has revealed properties that are highly unusual for a heme peroxidase but which may be relevant to its biological function
It is shown that the presence of Ca2؉ gives rise to biphasic kinetics for compound I formation, with a slow phase as described above and a fast phase that exhibits a second order rate constant more typical of a classical peroxidase (k1app ؍1.5 ؋ 107 M؊1 s؊1, which is pH-independent between 3.3 and 5.0)
The association rate constant is strongly dependent on pH and greatly favored at low pH, which indicates that the Kinetically Defined Forms of BP 1—Scheme I gives a simple overview of kinetically defined forms of BP 1 and the reaction characteristics of these forms with hydrogen peroxide
Summary
Materials—BP 1 was purified from barley grain [19] and stored at 4 °C as an ammonium sulfate precipitate or in freeze-dried form. The effects of calcium and chloride ions on the reactions of BP 1 and hydrogen peroxide or fluoride were examined in 50 mM sodium citrate buffer without CaCl2 (with and without EDTA) and with CaCl2. Titration with CaCl2 in citrate buffer were performed by adding aliquots of buffered NaCl or CaCl2 to the enzyme and monitored by absorption spectrometry. The effects of ionic strength were examined in 50 mM sodium citrate buffer, pH 3.97, in the presence of 1 mM CaCl2 at 0.04 M ionic strength and after adding K2SO4 or Na2SO4 to 0.5 M ionic strength. The effect of the ionic strength on the BP 1 absorption spectra in the absence and presence of 1 mM CaCl2 was examined at pH 3.25, 3.97, and 4.93 at 0.04, 0.1, and 0.5 M ionic strength using the same buffers. Experimental conditions are elaborated in the figure and table legends
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
