Effect of pH upon the reaction kinetics of the enzyme-substrate compounds of catalase.

The existence of acid-linked groups in hemoproteins may be observed by studies of the effect of pH upon the nature of the enzyme and its compounds or upon the speed with which the enzyme compounds are formed. Since the activity of enzymatically active hemoproteins depends upon compound formation with the substrate, the effect of pH upon the overall activity may also demonstrate heme linkages. Both of these approaches have been used by Theorell and Agner (1, 2) in their studies of catalase and have resulted in their postulation of a heme-linked hydroxyl group of pK 3.8 bound to catalase hematin, although they were unable to show a direct effect of this heme linkage upon catalase activity. In this paper new and rapid methods have been used for measuring the effect of pH upon catalase activity, not only in the decomposition of hydrogen peroxide but also in the oxidation of alcohol, formic acid, and nitrous acid. Studies have also been made of the effect of pH upon the kinetics of the catalase-peroxide compounds: their speed of formation, their speed of transition from one type to the other, and their speed of “spontaneous” decomposition into the free enzyme. It is found that all of these reactions are pH-insensitive in the range 5 to 9. Below pH 5 both the transition and the “spontaneous” decomposition reactions are accelerated, the latter in direct proportion to the hydrogen ion concentration, suggestive of a kinetically operative heme linkage. Below pH 4 the activity towards hydrogen peroxide diminishes, but not rapidly enough to correspond to pK 3.8 for Agner and Theorell’s heme-linked hydroxyl group (2). In the alkaline region this over-all. activity falls off below pH 9, owing possibly to a new heme linkage in catalase. The reaction of catalase hydrogen peroxide with alcohols exhibits remarkable pH stability; negligible change is measured from pH 4.3 to 12.0. It is shown here that this complex reacts only with the undissociated molecules of nitrous and formic acids. MethodsThe velocity constant for the reaction of catalase with hydrogen peroxide may be measured indirectly by a study of the effect of