HISTIDINE TO ALANINE MUTANTS OF HUMAN DIHYDROOROTATE DEHYDROGENASE - IDENTIFICATION OF A BREQUINAR-RESISTANT MUTANT ENZYME

Dihydroorotate dehydrogenase (DHODase) is the rate-limiting enzyme of the mammalian de novo pyrimidine biosynthesis pathway, and is the mole cular target of the antiproliferative, immunosuppressive compound breq uinar sodium (BQR). We have shown previously that the activity of the recombinant human enzyme displays pH and diethylpyrocarbonate sensitiv ities that implicate a critical role for one or more histidine residue s in catalysis [Copeland et al., Arch Biochem Biophys 323: 79-86, 1995 .]. Here we report the results of alanine scanning mutagenesis for eac h of the 8 histidine residues of the recombinant human enzyme. In most cases, the replacement of histidine by alanine had little effect on t he K-m values of the two substrates, dihydroorotate and ubiquinone, or on the overall k(cat) of the enzymatic reaction. Replacement of H71, H129, and H364 by alanine, however, completely abolished enzymatic act ivity. The loss of activity for the H71A mutant was unexpected, since this residue is not conserved in the homologous rat enzyme; in the rod ent enzyme this residue is an asparagine. Replacement of H71 by aspara gine in the human enzyme led to a full recovery of enzymatic activity, indicating that a histidine is not required at this position. Replace ment of H26 by alanine led to about a 10-fold reduction in catalytic a ctivity relative to the wild-type enzyme, with no significant perturba tion of the substrate K-m values. This mutant was, however, at least 1 67-fold less sensitive to inhibition by the noncompetitive inhibitor B QR. While the wild-type and other mutant enzymes displayed IC50 values for BQR inhibition between 6 and 10 nM, the H26A mutant was inhibited less than 25% at concentrations of BQR as high as 150 nM. These data suggest that H26 plays an important role in BQR binding to the enzyme. (C) 1997 EIsevier Science Inc.