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Role of lys100 in human dihydroorotate dehydrogenase: mutagenesis studies and chemical rescue by external amines.

Abstract：

: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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Jiang W,Locke G,Harpel MR,Copeland RA,Marcinkeviciene J

doi

10.1021/bi000630d

subject

Has Abstract

pub_date

2000-07-11 00:00:00

pages

7990-7

issue

27

eissn

0006-2960

issn

1520-4995

pii

bi000630d

journal_volume

39

pub_type

杂志文章

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