ISSN:
1432-1327
Schlagwort(e):
Key words Peroxidase
;
Protein engineering
;
Distal residues
;
Compound I formation
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
Abstract Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k 1 = 1.4×102 M–1s–1) compared with both native-glycosylated and recombinant forms of HRPC (k 1 = 1.7×107 M–1s–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k 1 = 1.1×104 M–1s–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k 2 = 142 s–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/s007750050032
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