Document Detail


Rescue of His-42 --> Ala horseradish peroxidase by a Phe-41 --> His mutation. Engineering of a surrogate catalytic histidine.
MedLine Citation:
PMID:  8798724     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of approximately 10(6) and of guaiacol oxidation by a factor of approximately 10(4). In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at approximately pH 6.3. Addition of H2O2 produces a Compound I species (lambdamax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (lambdamax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 x 10(4) M-1 s-1. This is to be compared with 0.9 x 10(7) M-1 s-1 for wild-type HRP and 19 M-1 s-1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s-1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s-1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.
Authors:
M I Savenkova; S L Newmyer; P R Montellano
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  271     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1996 Oct 
Date Detail:
Created Date:  1996-11-25     Completed Date:  1996-11-25     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  24598-603     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
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MeSH Terms
Descriptor/Qualifier:
Alanine / genetics*
Animals
Cell Line
Histidine / genetics*
Horseradish Peroxidase / genetics,  isolation & purification,  metabolism*
Kinetics
Mixed Function Oxygenases / metabolism
Mutagenesis, Site-Directed
Oxidation-Reduction
Phenylalanine / genetics*
Recombinant Proteins / genetics,  isolation & purification,  metabolism
Spodoptera
Grant Support
ID/Acronym/Agency:
GM32488/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Recombinant Proteins; 56-41-7/Alanine; 63-91-2/Phenylalanine; 71-00-1/Histidine; EC 1.-/Mixed Function Oxygenases; EC 1.11.1.-/Horseradish Peroxidase; EC 1.14.-/peroxygenase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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