hydroxy-L-arginine to citrulline and nitric oxide. The homodimeric enzyme contains one heme/monomer, and that cofactor is thought to mediate both partial reactions. Here we show by electron paramagnetic resonance spectroscopy that binding of substrate L-arginine to neuronal NOS perturbs the heme cofactor binding pocket without directly interacting as a sixth axial heme ligand; heme iron is exclusively high spin. In contrast, binding of L-thiocitrulline, a NOS inhibitor, produces both high and low spin iron spectra; L-thiocitrulline sulfur is a sixth axial heme ligand in one, but not all, of the low spin forms. The high spin forms of the L-thiocitrulline NOS complex display a distortion in the opposite direction to that caused by L-arginine binding. The findings elucidate the binding interactions of L-arginine and L-thiocitrulline to neuronal NOS and demonstrate that each causes a unique perturbation to the heme cofactor pocket of NOS.
Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide and L-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of the L-arginine-binding site. We report here studies with L-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the -NH 2 , ؍NH, or ؍NH 2 ؉ groups of L-arginine. Replacement groups include -NH-alkyl, -alkyl, ؍O, and ؍S. Binding of L-canavanine, an analog unable to form hydrogen bonds involving a N 5 -proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the L-arginine-binding site and use IC 50 or K i values and optical difference spectra to quantitate their affinity relative to L-arginine. We find that the non-reactive guanidinium nitrogen of L-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3-6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design. Nitric-oxide synthase (NOS)1 catalyzes the two-step oxidation of L-arginine to L-citrulline and nitric oxide (NO). Oxygen and NADPH are co-substrates, and N -hydroxy-L-arginine (NOH-Arg) is a tightly bound intermediate (1, 2). The enzyme is active as a homodimer, and each monomer is comprised of a heme-and tetrahydrobiopterin-containing oxygenase domain that binds and oxidizes L-arginine and a FAD-and FMNcontaining reductase domain that delivers electrons from NADPH to heme. Once reduced, the heme cofactor binds and activates O 2 , which in turn reacts with a terminal guanidinium nitrogen of the substrate L-arginine that is bound ϳ4 Å from the heme iron (3). That reactive, "proximal" nitrogen is first hydroxylated, forming NOH-Arg, and then oxidized further to NO. The other, previously equivalent guanidinium nitrogen is bound farther away from the heme cofactor and does not react; that "distal" nitrogen becomes the terminal -NH 2 group of the product L-citrulline.There are three major isoforms of NOS in mammals (1, 2, 4). Two constitutive, Ca 2ϩ /calmodulin-regulated isoforms were initially identified in neurons (nNOS) and vascular endothelial cells (eNOS). An inducible, transcriptionally regulated isoform (iNOS) was initially identified in macrophages but can be expressed in response to inflammatory cytokines and endotoxin in many cell types. Neuronal NOS has a role in neurotransmission and/or neuromodulation (5, 6), whereas eNOS produces NO that has an important role in controlling vasorelaxation and blood pressure (7,8). Nitric oxide derived from iNOS plays ...
Movies and movie clips have been used by many instructors to teach chemistry. Entire movies based on true chemical stories are used because they provide students with a common experience after which instructors can launch writing lessons about the chemistry, the scientists, or engineers, or even postscripts to the story presented in the film. In contrast, movie clips are used to animate a chemical topic during lecture in a way that grabs student attention. This gives students a strong anchor upon which they can contextualize the rest of the lesson. To find the most pedagogically useful clips, we formulated a hypothesis that clips with popular actors, incredible sets, memorable dialog, and special chemical effects would be the most useful for instruction because they would have the strongest anchoring capacity. That is, clips with more Wow! should be more useful for teaching and learning. The results of our study establish a set of criteria for choosing clips from feature films that chemistry instructors can use to grab the student attention and maximize learning.
SummaryThe study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences. To help understand which primase functions were conserved, primase and its replication partner helicase from the pathogenic Gram-positive bacteria Staphylococcus aureus were compared in detail with that of E. coli primase and helicase. The conserved properties were to primer initiation and elongation and included slow kinetics, low fidelity and poor sugar specificity. The significant differences included S. aureus primase having sixfold higher kinetic affinity for its template than E. coli primase under equivalent conditions. This naturally higher activity was balanced by its fourfold lower stimulation by its replication fork helicase compared with E. coli primase. The most significant difference between the two primases was that S. aureus helicase stimulation did not broaden the S. aureus primase initiation specificity, which has important biological implications.
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