Linked Life Data

10891080

Source:http://linkedlifedata.com/resource/pubmed/id/10891080

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
27
pubmed:dateCreated
2000-8-4
pubmed:abstractText
Chemical modification, mutagenesis, chemical rescue, and isotope effect studies are used to identify and probe the roles of several conserved amino acid groups in catalysis by human dihydroorotate dehydrogenase. Time- and pH-dependent inactivation of human dihydroorotate dehydrogenase by trinitrobenzenesulfonate implicates at least one critical lysyl residue in catalysis. Of four highly conserved lysines, only the cognate of Lys255 was previously suggested to have catalytic functionality. We now show that replacement of either Lys184 or Lys186 by mutagenesis does not impact, whereas substitution of Lys100 abolishes, enzymatic activity. However, activity is partially restored to K100C (or K100A) by inclusion of exogenous primary amines in reaction mixtures. This rescued activity saturates with respect to numerous amines and exhibits a steric discrimination reflected in K(d,(amine)) values. For all amines, rescued k(cat) values were only approximately 10% of wild type and independent of amine basicity. K(M) values for dihydroorotate and coenzyme Q(0) were similar to wild type. Thus, exogenous amines (as surrogates for Lys100) apparently complement a chemical, not binding, step(s) of catalysis, which does not entail proton transfer. In support of this postulate, solvent kinetic isotope effect analysis indicates that Lys100 stabilizes developing negative charge on the isoalloxazine ring of flavin mononucleotide during hydride transfer, as has been observed for a number of flavoprotein oxidoreductases. Ser215 of human dihydroarotate dehydrogenase (DHODase) was also studied because of its alignment with the putative active-site base Cys130 of Lactococcus lactisDHODase. Substantial retention of activity by S215C, yet complete loss of activity for S215A, is consistent with Ser215 serving as the active-site base in the human enzyme.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
11
pubmed:volume
39
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7990-7
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
Role of lys100 in human dihydroorotate dehydrogenase: mutagenesis studies and chemical rescue by external amines.
pubmed:affiliation
Department of Chemical Enzymology, DuPont Pharmaceuticals Company, P. O. Box 80400, Wilmington, Delaware 19880-0400, USA.
pubmed:publicationType
Journal Article