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Chimeras of Nitric Oxide Synthase Engineered to Have Distinct Kinetic Parameters and Product Profiles
Author(s) -
Binder Katherine Emoke,
Wang ZhiQiang,
Haque Mohammad Mahfuzul,
Stuehr Dennis J.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.890.8
Subject(s) - heme , chemistry , nitric oxide , nitric oxide synthase , catalysis , ferric , hemeprotein , dissociation (chemistry) , ferrous , chimera (genetics) , catalytic cycle , biophysics , biochemistry , enzyme , inorganic chemistry , biology , organic chemistry , gene
The three Nitric Oxide Synthases (NOSs) play vital roles in biological processes through their synthesis of nitric oxide (NO). According to our catalytic model, three parameters determine the catalytic activity and behavior of any mammalian NOS: heme reduction, ferrous‐NO oxidation and its NO dissociation rate from the ferric heme‐NO complex. These parameters vary significantly among the three NOSs, and cause each NOS to distribute differently between an NO‐releasing cycle and an NO dioxygenase cycle during catalysis. We have investigated if NOS changes in a predictable way when the kinetic parameters vary from their natural ranges. We examined how modulating heme reduction rate and a slow ferric‐NO dissociation rate influences NOS catalytic behavior. To accomplish this, we have constructed four NOS chimeras. Two iNOS‐nNOS chimera proteins (iNOS‐nNOS and iNOS‐nNOS S1412D) were constructed to have non‐native heme reduction rates. These chimeras were found to have a fast rate of heme reduction. In addition, we have generated two other NOS chimeras (V346IiNOS‐nNOS and the V346IiNOS‐nNOSS1412D). These two chimeras were found to have a moderate rate of heme reduction and a slower than normal ferric‐NO dissociation rate. How the measured changes in kinetic parameters correlate with measured NO release and NO dioxygenase activities of each chimera will be discussed. Support by NIH CA53914.