Nitric Oxide Synthase: Difference between revisions
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<applet load='2g6h' size='300' color='white' frame='true' align='right' caption='Oxygenase domain' /> | <applet load='2g6h' size='300' color='white' frame='true' align='right' caption='Oxygenase domain' /> | ||
The <scene name='Sandbox_5/Nos_oxygenase_med_cofaktore/3'>oxygenase domain</scene> contains the active site of the enzyme. The active site binds the substrate <scene name='Nitric_oxide_synthase/Nos_oxygenase_arg/3'>L-Arginine</scene> ([http://en.wikipedia.org/wiki/Arginine Arginine]) which is converted into citruline and NO (explained in details below). The domain has three cofactors bound: | The <scene name='Sandbox_5/Nos_oxygenase_med_cofaktore/3'>oxygenase domain</scene>(PDB:2G6H) contains the active site of the enzyme. The active site binds the substrate <scene name='Nitric_oxide_synthase/Nos_oxygenase_arg/3'>L-Arginine</scene> ([http://en.wikipedia.org/wiki/Arginine Arginine]) which is converted into citruline and NO (explained in details below). The domain has three cofactors bound: | ||
<scene name='Nitric_oxide_synthase/Nos_oxygenase_bh4/1'>H4B</scene> ([http://en.wikipedia.org/wiki/BH4 (6R).5,6,7,8-Tetrahydrobiopterin]) | <scene name='Nitric_oxide_synthase/Nos_oxygenase_bh4/1'>H4B</scene> ([http://en.wikipedia.org/wiki/BH4 (6R).5,6,7,8-Tetrahydrobiopterin])(PDB:2G6H) | ||
<scene name='Nitric_oxide_synthase/Nos_oxygenase_heme/1'>Heme</scene> ([http://en.wikipedia.org/wiki/Heme Heme]) | <scene name='Nitric_oxide_synthase/Nos_oxygenase_heme/1'>Heme</scene> ([http://en.wikipedia.org/wiki/Heme Heme])(PDB:2G6H) | ||
<scene name='Nitric_oxide_synthase/Nos_oxygenase_zinc/4'>Zinc ion</scene> | <scene name='Nitric_oxide_synthase/Nos_oxygenase_zinc/4'>Zinc ion</scene>(PDB:2G6H) | ||
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==== Substrate binding ==== | ==== Substrate binding ==== | ||
The active site is highly conserved in the different NOS species. Thus it is possible to discuss substrate binding i general terms. The NOS enzyme binds its substrate (L-arginine) in the distal pocket by hydrogen bindings both to the guanidino[http://en.wikipedia.org/wiki/Guanidino] end and the amino acid end. <scene name='Nitric_oxide_synthase/Substratebinding_test/1'>Substrate in the catalytic site</scene> is shown in green with the heme and H<sub>4</sub>B shown. NOS binds its substrate by coordinating CO(or O<sub>2</sub>) to the heme at the site occupied by oxygen<ref>PMID:9376373 </ref>(it is the opposite site of the Cys coordination to heme - look in the 'heme' section). The binding of substrate leads to a 2-step transformation first to N-hydroxy-L-arginine (the tightly bound intermideate) and then NO and L-Citrulline. The product NO can then either diffuse out of the <scene name='Nitric_oxide_synthase/Substratebinding_distal_pocket/1'>cavity</scene> or bind to the heme and function in NO auto-inhibition though this inhibition is diverse throughout the 3 isoforms<ref>PMID:15598509</ref>. | The active site is highly conserved in the different NOS species. Thus it is possible to discuss substrate binding i general terms. The NOS enzyme binds its substrate (L-arginine) in the distal pocket by hydrogen bindings both to the guanidino[http://en.wikipedia.org/wiki/Guanidino] end and the amino acid end. <scene name='Nitric_oxide_synthase/Substratebinding_test/1'>Substrate in the catalytic site</scene> is shown in green with the heme and H<sub>4</sub>B shown. NOS binds its substrate by coordinating CO(or O<sub>2</sub>) to the heme at the site occupied by oxygen<ref>PMID:9376373 </ref>(it is the opposite site of the Cys coordination to heme - look in the 'heme' section). The binding of substrate leads to a 2-step transformation first to N-hydroxy-L-arginine (the tightly bound intermideate) and then NO and L-Citrulline. The product NO can then either diffuse out of the <scene name='Nitric_oxide_synthase/Substratebinding_distal_pocket/1'>cavity</scene>(PDB:2G6H) or bind to the heme and function in NO auto-inhibition though this inhibition is diverse throughout the 3 isoforms<ref>PMID:15598509</ref>. | ||
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<applet load='2g6h' size='300' frame='true' align='right' caption='Tetrahydrobiopterin' /> | <applet load='2g6h' size='300' frame='true' align='right' caption='Tetrahydrobiopterin' /> | ||
<scene name='Sandbox_5/Nos_oxygenase_bh4/11'>H4B</scene> is a cofactor. NOS contains two molecules of <scene name='Sandbox_5/Begge_h4b/1'>H4B</scene>, one in each monomer. The active center forms a kind of <scene name='Nitric_oxide_synthase/Substratebinding_distal_pocket/2'>tunnel</scene>, because of the dimeric structure. This gives H<sub>4</sub>B the opportunity to play a big role in the control of subunit interactions and active-center formation. H<sub>4</sub>B therefor is more of a structurel cofactor, in that it keeps the dimer stabilized by integration in to the hydrophobic parts of the dimer. Here it helps substrate interactions by lining the active-center channel and hydrogen bonding to the heme propionate amd to alfa7 which is two elements involved in L-Arg binding. Its structural importense is also reconned to play a role in dimer formation, and major conformational changes leading to the formation of the active site channelform<ref>PMID:9875848</ref>. | <scene name='Sandbox_5/Nos_oxygenase_bh4/11'>H4B</scene>(PDB:2G6H) is a cofactor. NOS contains two molecules of <scene name='Sandbox_5/Begge_h4b/1'>H4B</scene>(PDB:2G6H), one in each monomer. The active center forms a kind of <scene name='Nitric_oxide_synthase/Substratebinding_distal_pocket/2'>tunnel</scene>(PDB:2G6H), because of the dimeric structure. This gives H<sub>4</sub>B the opportunity to play a big role in the control of subunit interactions and active-center formation. H<sub>4</sub>B therefor is more of a structurel cofactor, in that it keeps the dimer stabilized by integration in to the hydrophobic parts of the dimer. Here it helps substrate interactions by lining the active-center channel and hydrogen bonding to the heme propionate amd to alfa7 which is two elements involved in L-Arg binding. Its structural importense is also reconned to play a role in dimer formation, and major conformational changes leading to the formation of the active site channelform<ref>PMID:9875848</ref>. | ||
[[image:H4b_hydrogenbindinger2.png|thumb|left|Hydrogenbondings in H<sub>4</sub>B binding site]] | [[image:H4b_hydrogenbindinger2.png|thumb|left|Hydrogenbondings in H<sub>4</sub>B binding site]] | ||
The H<sub>4</sub>B is bound by hydrogen-bonds to several of the molekules surrounding it, including the substrate L-Arg. The substrate is H-bonded to the 4-keto group of pterin, and to one of the heme propionate groups, that has two carboxylate oxygens in use for H-bonds. These oxygens are further H-bonded to the 4-keto group of pterin, through water, and directly to N(3) and NH<sub>2</sub> on C (2)<ref>PMID:9875848</ref>. The big picture of all the H-bonds can be seen by clicking on the figure on the left. [[image:mette.png|thumb|right|model for NOS oxygen activation]] | The H<sub>4</sub>B is bound by hydrogen-bonds to several of the molekules surrounding it, including the substrate L-Arg. The substrate is H-bonded to the 4-keto group of pterin, and to one of the heme propionate groups, that has two carboxylate oxygens in use for H-bonds. These oxygens are further H-bonded to the 4-keto group of pterin, through water, and directly to N(3) and NH<sub>2</sub> on C (2)<ref>PMID:9875848</ref>. The big picture of all the H-bonds can be seen by clicking on the figure on the left. [[image:mette.png|thumb|right|model for NOS oxygen activation]] | ||
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===Zinc=== | ===Zinc=== | ||
In order for Nos to be active it has to dimerize and bind H<sub>4</sub>B. The two monomers are held together by a single structural <applet load='2g6h' size='300' frame='true' align='right' caption='Zinc' /><scene name='Nitric_oxide_synthase/Zink/2'>zinc ion</scene> | In order for Nos to be active it has to dimerize and bind H<sub>4</sub>B. The two monomers are held together by a single structural <applet load='2g6h' size='300' frame='true' align='right' caption='Zinc' /><scene name='Nitric_oxide_synthase/Zink/2'>zinc ion</scene>(PDB:2G6H) | ||
which is situated at the interface of the dimer.<ref>PMID: 10074942</ref> The zinc ion is tetrahedrally coordinated and has four cysteins bound as ligands (two from each monomer - Cys109 and Cys104). Further, it is found that zinc binds together the oxygenase domains of the monomers. The zinc ion is found at a region which connects the N-terminal hook and the subunit core. The coordination of zinc arranges the N-terminal hooks so that they interact with their own subunit. However, when there is no zinc ion present, two of the thiolate | which is situated at the interface of the dimer.<ref>PMID: 10074942</ref> The zinc ion is tetrahedrally coordinated and has four cysteins bound as ligands (two from each monomer - Cys109 and Cys104). Further, it is found that zinc binds together the oxygenase domains of the monomers. The zinc ion is found at a region which connects the N-terminal hook and the subunit core. The coordination of zinc arranges the N-terminal hooks so that they interact with their own subunit. However, when there is no zinc ion present, two of the thiolate | ||
ligands (cysteines) form a disulfide bond connecting the two subunits<ref>PMID: 10562539</ref>. | ligands (cysteines) form a disulfide bond connecting the two subunits<ref>PMID: 10562539</ref>. | ||
