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|Title:||The catalytic mechanism of ascorbate peroxidase : probing the effects of changes in enzyme and substrate structure|
|Presented at:||University of Leicester|
|Abstract:||Recombinant pea cytosolic ascorbate peroxidase (rAPX) has been isolated and the mechanistic properties have been investigated. Rate constants for formation of compound I with H2O2 and other organic peroxides were measured. The data indicate that the structure and size of peroxide dictate the rate of compound I formation. Rate constants for reduction of compound I and compound II were measured with L-ascorbic acid and other derivatised forms of ascorbate. Reduction of compound II showed evidence for formation of an enzyme-substrate complex. The rate constants for compound II reduction, by the various ascorbate-based substrates, were controlled by the thermodynamic driving force of the reaction.;Variants H42A and H42E were constructed to investigate the catalytic role of His42 in rAPX catalysis. The observed pseudo-first-order rate constant for the reaction between the His42 variants and hydrogen peroxide saturates at high peroxide concentration. The data are consistent with a two-step mechanism involving the formation of an APX-H2O2 intermediate whose conversion to compound I is rate-limiting. pH-Dependence studies on compound I formation reveal His42 as the key ionizable residue. Rapid photodiode array spectrophotometry revealed the presence of a transient intermediate for H42A, with a spectrum consistent with a ferric-hydroperoxy complex. The rate of formation of compound I and peroxidase activity of H42E were significantly greater than H42A, however, addition of exogenous imidazoles partially rescues both the rate of compound I formation and peroxidase activity for H42A.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Chemistry|
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