Linked Life Data

9298967

Source:http://linkedlifedata.com/resource/pubmed/id/9298967

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
38
pubmed:dateCreated
1997-10-16
pubmed:abstractText
The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
23
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
11472-8
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone.
pubmed:affiliation
Department of Chemistry, University of California, Berkeley, California 94720, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.