Putative hydrogen bond network in the heme distal site of horseradish peroxidase.
The N delta 1 atom of the distal His of several peroxidases is known to make a hydrogen bond with the side chain oxygen of Asn. Thus, a mutant horseradish peroxidase, in which Asn70 is replaced by Val, has been expressed in Esherichia coli to disrupt the putative hydrogen bond. Substitution of Asn70 to Val reduces the rate constant for the compound I formation from 1.6 x 10(7) (native) to 6 x 10(5) M-1s-1. The rate constant for reduction of compound I of N70V by guaiacol has been also reduced from 7.8 x 10(6) (native) to 1.2 x 10(5) M-1s-1. While compound I of N70V is stable and reduced to the resting state of the mutant without apparent formation of compound II at neutral pH, compound II of N70V is obtained as a stable intermediate at alkaline pH. Similar alteration of the reactivity has been observed in the reaction with guaiacol.
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
S
S
Nagano
M
M
Tanaka
Y
Y
Watanabe
I
I
Morishima
D000595:Amino Acid Sequence; D001216:Asparagine; D003001:Cloning, Molecular; D003576:Electron Transport Complex IV / Q000737:chemistry; D004926:Escherichia coli; D006418:Heme; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014633:Valine
D011994:Recombinant Proteins; D006418:Heme; D001216:Asparagine; D006735:Horseradish Peroxidase; D003576:Electron Transport Complex IV; D014633:Valine