Abstract：
:In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221-226] that the active-site His10 of NADH peroxidase functions as an essential acid-base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The k(cat) values for both H10Q and H10A peroxidases, and the pH profile for k(cat) with H10Q, are very similar to those observed with wild-type peroxidase. Both mutants, however, exhibit K(m)(H2O2) values much higher (50-70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E.NADH intermediate in the conversion of E-->EH2 is confirmed by diode-array spectral analyses with H10A. Both H10Q and H10A mutants, in their oxidized E(FAD, Cys42-sulfenic acid) forms, exhibit enhanced long-wavelength absorbance bands (lambda(max) = 650 nm and 550 nm, respectively), which most likely reflect perturbations in a charge-transfer interaction between the Cys42-sulfenic acid and FAD. Combined with the 50-fold increase in the second-order rate constant for H2O2 inactivation (via Cys42-sulfenic acid oxidation) of the H10Q mutant, these observations support the proposal that His10 functions in part to stabilize the unusual Cys42-sulfenic acid redox center within the active-site environment.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Crane EJ 3rd,Parsonage D,Claiborne Adoi
10.1021/bi952347ysubject
Has Abstractpub_date
1996-02-20 00:00:00pages
2380-7issue
7eissn
0006-2960issn
1520-4995pii
bi952347yjournal_volume
35pub_type
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