Abstract
Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.
Highlights
Elementary reactions have been studied quantita- iluminescence can be observed
Conversion of native HRP to compound I is caused by the peracid autoxidation product of IBAL or by H202added to the reaction mixture [15].The rate constantfor the reaction of the native enzyme with H202is 1.8X lo7M" s" [25] andwith an organic peracid (p-nitroperbenzoic acid) is 3.0 X lo7 M" s-' [26]
The rate constant for the reaction between the native enzyme and the peracid derived from IBAL should be of the same order of magnitude (k2 lo7M" s-')
Summary
Elementary reactions have been studied quantita- iluminescence can be observed. This work is concerned with tivelyinthe complex overall processcatalyzedby the IBAL/02/HRP system which generates acetone in the horseradish peroxidase whereby isobutyraldehyde andtriplet state andformic acid. A mechanism for this molecular oxygen react to form triplet state acetone reaction was proposed and qualitative aspectswere described and formic acid. The hydes appears to react to unionized phenols with burst is caused by the followinsgequence: 1)an initial compounds I and I1of HRP and a common mechanism is high yield of compound I is formed from reaction of proposed. After this burst a steady state phase is observed .in phate derivatives from IBAL are more efficient catalysts of which the rate-limiting step is the conversion of the enolization than phosphate butarenotsubstrates for the keto to the enol form of the aldehyde catalyzed by enzyme [16]. Both the initial burstof light and the steady staetme ission from triplet acetone can be observed with the naked eye
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