Catalytic Activities and Structural Properties of Horseradish Peroxidase Distal His42 → Glu or Gln Mutant

Abstract

The distal histidine (His) is highly conserved in peroxidases and has been considered to play a major role as a general acid-base catalyst for peroxidase reaction cycle. Recently, however, the X-ray structure of chloroperoxidase from the marine fungus Caldariomyces fumago has revealed that a glutamic acid is located at the position where most of the peroxidase has a histidine residue, suggesting that the carboxyl group in the glutamic acid (Glu) can also assist cleavage of an O-O bond in peroxides [Sundaramoorthy, M., Terner, J., & Poulos, T. L. (1995) Structure 3, 1367-1377]. In order to investigate catalytic roles of the glutamic acid at the distal cavity, two horseradish peroxidase mutants were prepared, in which the distal His42 has been replaced by Glu (H42E) or Gln (H42Q). The formation rate of compound I in the H42E mutant was significantly greater than that for the H42Q mutant, indicating that the distal Glu can play a role as a general acid-base catalyst. However, the peroxidase activity of the H42E mutant was still lower, compared to that for native enzyme. On the basis of the CD, resonance Raman, and EPR spectra, it was suggested that the basicity of the distal Glu is lower than that of the distal His and the position of the distal Glu is not fixed at the optimal position as a catalytic amino acid residue, although no prominent structural changes around heme environment were detected. The less basicity and improper positioning of the distal Glu would destabilize the heme-H2O2-distal Glu ternary intermediate for the peroxidase reaction. Another characteristic feature in the mutants was the enhancement of the peroxygenase activity. Since the peroxygenase activity was remarkably enhanced in the H42E mutant, the distal Glu is also crucial to facilitate the peroxygenase activity as well as the enlarged distal cavity caused by the amino acid substitution. These observations indicate that the distal amino acid residue is essential for function of peroxidases and subtle conformational changes around the distal cavity would control the catalytic reactions in peroxidase.

Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

“…The k 2 values for guaiacol oxidation are within the same order of magnitude as the published value for the wild-type enzyme (25,32), although the value for the commercial enzyme measured here was 10 -30% lower than that in the literature. The values recorded for the wild-type enzyme before and after treatment are consistent with the ones obtained for the commercial enzyme, and show no significant influence of the treatment with peroxide.…”

“…The failed mutants show, as might be expected, that the introduction of a negatively charged carboxylic acid residue into the active site can cause serious structural perturbations. The introduction of a carboxylate into the active site of HRP has been achieved earlier for other purposes, notably the substitution of His 42 by a glutamate in efforts to prepare a chloroperoxidase mimic (29,32,36) or to disrupt the active site hydrogen-bonding network associated with a bound benzhydroxamic acid molecule (37). Asn 70 , which interacts with His 42 , has also been successfully replaced by a glutamate (17,38,39).…”

“…The S73E mutant for its part gave values approximately 3 times lower than the wild type, and this was not significantly changed by the oxidative treatment. The instability of compound II precludes the measurements of k 3 by stopped flow kinetics under the conditions used, so we tried to obtain k 3 values through steady state kinetic measurements as described elsewhere (25,32,33). Unfortunately, in our hands the model did not permit the calculation of reliable values by this method.…”

Effects of mutations in the helix G region of horseradish peroxidase

“…There are a few reports of serendipitously altered catalytic properties, such as peroxygenase activity ( [2]; His42→Glu), thioanisole sulfoxidation ( [3]; His42→Ala/Phe41→His), or both ([4]; Phe41→Leu). There are also reports of altered kinetic properties arising from single substitutions: for example, an increased V m /E ratio for ABTS resulted from a single Ser35→Lys substitution [5], whilst a Asn70→Asp substitution led to decreased guaiacol oxidation rates [6].…”

Catalytic peroxidation of nitrogen monoxide and peroxynitrite by globins

“…1), which significantly lowers the pK a values of catalytic His and Arg distal residues (66,67). This idea is further strengthened by the following: (i) the evidence that site-directed mutants of horse heart Mb (Thr39Ile, Lys45Asp, Phe46Leu, and Ile107Phe) and sperm whale Mb (Thr67Arg and Thr67Arg/ Ser92Asp) display a significant increase of the peroxidase activity (25,68,69), and (ii) site-directed mutants of cytochrome c peroxidase (His175Gln, His175Glu, and His175Cys) and horseradish peroxidase (Arg38Leu, His42Glu, His42Gln) show a substantial decrease of the peroxidase activity (70)(71)(72).…”