The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases.
Kinetic parameters and primary deuterium isotope effects have been determined for wild-type dihydropteridine reductase (EC 1.6.99.7) and the Ala133Ser, Lys150Gln, Tyr146His, Tyr146Phe single, and Tyr146Phe/Ala133Ser and Tyr146Phe/Lys150Gln double mutant enzyme forms using the natural substrate, quinonoid (6R)-l-erythro-dihydrobiopterin (qBH2) and an alternate substrate, quinonoid 6,7-dimethyldihydropteridine (q-6,7-diMePtH2). Mutation at either Tyr146 or Lys150 resulted in pronounced changes in kinetic parameters and isotope effects for both pterin substrates, confirming a critical role for these residues in enzyme-mediated hydride transfer. By contrast, the Ala133Ser mutant was practically indistinguishable from wild-type enzyme. The changes observed, however, were quite different for the two pterin substrates. Thus, kcat for q-6,7-diMePtH2 decreased across the series of mutants from a value of 150 s-1 for wild-type enzyme to essentially zero activity for the Tyr146Phe/Lys150Gln double mutant. Conversely, kcat for qBH2 increased 3-11-fold across the same series of mutants from the wild-type value of 23 s-1. For both pterin substrates, the Km (KPt) increased several orders of magnitude upon mutation of Tyr146 or Lys150, with the greater relative increase using qBH2. Significant primary deuterium isotope effects on kcat (Dkcat) and kcat/KPt (D(kcat/KPt)) observed for the Tyr146 and Lys150 mutants varied depending on the pterin substrate used and ranged up to a maximum value of 5.5-6. For qBH2, where Dkcat < Dkcat/KPt was consistently observed, the rate determining step is ascribed to release of the tetrahydropterin product. For q-6,7-diMePtH2, where in all cases Dkcat = Dkcat/KPt, catalysis is probably limited by an isomerization step occurring prior to hydride transfer. Modeling studies in which qBH2 was docked into the binary E:NADH complex provide a structural rationale for the observed differences between the two pterin substrates. The natural substrate, qBH2, 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 Asn186, Ser189, and Met190. The location of this loop within the three-dimensional structure is consistent with putative substrate binding loops for other members of the short chain dehydrogenase/reductase (SDR) family, which includes dihydropteridine reductase.
Kiefer
P M
PM
University of California at San Diego, La Jolla, California 92093, USA.
Grimshaw
C E
CE
Whiteley
J M
JM
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D004093:Dihydropteridine Reductase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011622:Pterins / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011622:Pterins; D011994:Recombinant Proteins; D001708:Biopterins; C017226:7,8-dihydrobiopterin; D004093:Dihydropteridine Reductase