Structural roles of the highly conserved glu residue in the heme distal site of peroxidases.
One of the highly conserved amino acid residues in the heme distal site of various fungal and plant peroxidases, glutamic acid 64 (Glu64) in horseradish peroxidase (HRP), interacts with a distal calcium ion through a hydrogen bond with a water molecule and its peptide carbonyl oxygen on the main-chain forms the hydrogen bond network to the distal His via the adjacent Asn residue, suggesting that the Glu residue is related to the stabilization of the calcium ion and catalytic activity of peroxidase [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., and Morishima, I. (1996) Biochemistry 35, 14251-14258]. To perturb the hydrogen bond with the adjacent Asn, we replaced the Glu with Pro (E64P) or Gly (E64G), which would alter the configuration of the main chain at position 64. Both of the mutants exhibited substantially depressed oxidation activities for hydroquinone and elementary reaction rates in the catalytic cycle. However, the E64S (Glu64 --> Ser) mutant, in which the configuration of the main chain and the hydrogen bond with Asn70 would not be affected but the interactions with the calcium ion are seriously perturbed by removal of the carboxylate, also showed quite low catalytic activity as observed for the E64P and E64G mutants. Spectral features for the E64S mutant are similar to those of the other mutants: the reorientation of the distal His, disruption of the hydrogen bond between the distal His and Asn70, and loss of the calcium ion. Thus, we can conclude that, in addition to forming the hydrogen bond network in the distal site, the Glu residue is a key residue for stable binding of the calcium ion, which maintains the structural integrity of the distal cavity, resulting in high peroxidase activity.
Tanaka
M
M
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan.
Ishimori
K
K
Morishima
I
I
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D011994:Recombinant Proteins; D018698:Glutamic Acid; D006418:Heme; D006735:Horseradish Peroxidase