Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.
The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.
Golser
Regina
R
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
Gorren
Antonius C F
AC
Mayer
Bernd
B
Schmidt
Kurt
K
D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D019281:Dimerization; D004727:Endothelium / Q000201:enzymology*; D015870:Gene Expression; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D020125:Mutation, Missense / Q000235:genetics*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010843:Pichia / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002147:Calmodulin; D003067:Coenzymes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III