Kinetic Studies on the Reaction of Compound II of Myeloperoxidase with Ascorbic Acid

نویسنده

  • Harold Van Wart
چکیده

Ascorbic acid is known to stimulate leukocyte functions. In a recent publication it was suggested that the role of ascorbic acid is to reduce compound II of myeloperoxidase back to the native enzyme (Bolscher, B. G. J. M., Zoutberg, G. R., Cuperus, R. A., and Wever, R. (1984) Biochim. Biophys. Acta 784, 189-191). In this paper we report rapid spectral scan and transient state kinetic results on the reaction of three myeloperoxidase compounds II, namely, human neutrophil myeloperoxidase, canine myeloperoxidase, and bovine spleen heme protein with ascorbate. We show by rapid scan spectra that compound II does not pass through any other intermediate when ascorbic acid reduces it back to native form. We also show that the reactions of all three compounds II involve a simple binding interaction before enzyme reduction with an apparent dissociation constant of 6.3 f 0.9 x 10S4 to 2.0 f 0.3 x 10m3 M and a first-order rate constant for reduction of 12.6 f 0.6 to 18.8 2 1.3 s-‘. The optimum pH is 4.5, and at this pH the activation energy for the reaction is 13.2 kJ mol-‘. Results of this work lend further evidence that the spleen green heme protein is very similar if not identical to leukocyte myeloperoxidase based on a comparison of spectral scans, pH-rate profiles, and kinetic parameters. We demonstrate that chloride cannot reduce compound II whereas iodide reduces compound II to native enzyme at a rate comparable to that of ascorbate. This explains why ascorbate accelerates chlorination but inhibits iodination. Formation of compound II is a dead end for the generation of hypochlorous acid; ascorbate regenerates more native enzyme to enhance the chlorination reaction namely: myeloperoxidase + peroxide + compound I followed by compound I + chloride + HOCl. On the other hand, ascorbate is a competitor with iodide for both compounds I and II and so inhibits iodination.

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تاریخ انتشار 2001