THE COMPARATIVE INTERACTION OF QUINONOID (6R)-DIHYDROBIOPTERIN AND ANALTERNATIVE DIHYDROPTERIN SUBSTRATE WITH WILD-TYPE AND MUTANT RAT DIHYDROPTERIDINE REDUCTASES

Kinetic parameters and primary deuterium isotope effects have been det ermined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala1 33Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the nat ural substrate, quinonoid (6R)-L-erythro-dihydrobiopterin (qBH(2)) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7 -diMePtH(2)). Mutation at either Tyr146 or Lys150 resulted in pronounc ed changes in kinetic parameters and isotope effects for both pterin s ubstrates, confirming a critical role for these residues in enzyme-med iated hydride transfer. By contrast, the Ala133Ser mutant was practica lly indistinguishable from wild-type enzyme, The changes observed, how ever, were quite different for the two pterin substrates. Thus, k(cat) for q-6,7-diMePtH(2) decreased across the series of mutants from a va lue of 150 s(-1) for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely, k(cat) for qBH(2) increased 3-11-fold across the same series of mutants from the wild-ty pe value of 23 s(-1). For both pterin substrates, the K-m (K-Pt) incre ased several orders of magnitude upon mutation of Tyr146 or Lys 150, w ith the greater relative increase using qBH(2). Significant primary de uterium isotope effects on k(cat) ((D)k(cat)) and k(cat)/K-Pt ((D)(k(c at)/K-Pt)) observed for Vie Tyr146 and Lps 150 mutants varied dependin g on the pterin substrate used and ranged up to a maximum value of 5.5 -6. For qBR(2), where (D)k(cat) < (D)k(cat)/K-Pt was consistently obse rved, the rate determining step is ascribed to release of the tetrahyd ropterin product. For q-6,7-diMePtH(2), where in all cases Dk(cat) = ( D)k(cat)/K-Pt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer, Modeling studies in which qBH(2) was docked into the binary E:NADH complex provide a structural rationa le fur the observed differences between the two pterin substrates. The natural substrate, qBH(2), displays a higher affinity for the enzyme active site, presumably due to interaction of the dihydroxypropyl side chain of the substrate with a polar loop of residues containing Asn18 6, Ser189, and Met190. The location of this loop within the three-dime nsional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) fam ily, which includes dihydropteridine reductase.