Linked Life Data

8652580

Source:http://linkedlifedata.com/resource/pubmed/id/8652580

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
7
pubmed:dateCreated
1996-7-30
pubmed:abstractText
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of NADH peroxidase functions as an essential acid-base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala.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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2380-7
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1996
pubmed:articleTitle
The active-site histidine-10 of enterococcal NADH peroxidase is not essential for catalytic activity.
pubmed:affiliation
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.