PMID: 8612649Mar 1, 1996Paper

Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine

European Journal of Biochemistry
D J GilfoyleA T Smith

Abstract

The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level...Continue Reading

References

May 1, 1995·Biochemical Society Transactions·P R Ortiz de MontellanoC Harmann
May 1, 1995·Biochemical Society Transactions·N C Veitch
May 1, 1995·Biochemical Society Transactions·G H LoewA T Smith
May 1, 1995·European Journal of Biochemistry·N C Veitch, R J Williams
Jul 1, 1993·Journal of Biotechnology·D Cai, M Tien

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Citations

Nov 27, 2002·Biotechnology and Bioengineering·Anne Marie O'BrienCiarán O'Fágáin
Sep 8, 2001·The Journal of Biological Chemistry·B D HowesG Smulevich
May 27, 2014·Glycobiology·Simona CaponeOliver Spadiut
Feb 23, 2019·International Journal of Molecular Sciences·Diana Humer, Oliver Spadiut

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