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 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.
Rasmussen
C B
CB
Department of Protein Chemistry, University of Copenhagen, Denmark.
Hiner
A N
AN
Smith
A T
AT
Welinder
K G
KG
D002118:Calcium / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D004492:Edetic Acid / Q000494:pharmacology; D005459:Fluorides / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008956:Models, Chemical; D010544:Peroxidases / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic
D007477:Ions; D010940:Plant Proteins; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D010544:Peroxidases; C070881:barley peroxidase; D005459:Fluorides; D002118:Calcium