1l0c
From Proteopedia
(New page: 200px<br /><applet load="1l0c" size="450" color="white" frame="true" align="right" spinBox="true" caption="1l0c, resolution 2.3Å" /> '''Investigation of the ...) |
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| - | [[Image:1l0c.jpg|left|200px]]<br /><applet load="1l0c" size=" | + | [[Image:1l0c.jpg|left|200px]]<br /><applet load="1l0c" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1l0c, resolution 2.3Å" /> | caption="1l0c, resolution 2.3Å" /> | ||
'''Investigation of the Roles of Catalytic Residues in Serotonin N-Acetyltransferase'''<br /> | '''Investigation of the Roles of Catalytic Residues in Serotonin N-Acetyltransferase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) | + | Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors. |
==About this Structure== | ==About this Structure== | ||
| - | 1L0C is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Ovis_aries Ovis aries] with COT as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Aralkylamine_N-acetyltransferase Aralkylamine N-acetyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.87 2.3.1.87] Full crystallographic information is available from [http:// | + | 1L0C is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Ovis_aries Ovis aries] with <scene name='pdbligand=COT:'>COT</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Aralkylamine_N-acetyltransferase Aralkylamine N-acetyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.3.1.87 2.3.1.87] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1L0C OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Ovis aries]] | [[Category: Ovis aries]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Angelis, J | + | [[Category: Angelis, J De.]] |
| - | [[Category: Burley, S | + | [[Category: Burley, S K.]] |
| - | [[Category: Cole, P | + | [[Category: Cole, P A.]] |
| - | [[Category: Scheibner, K | + | [[Category: Scheibner, K A.]] |
[[Category: COT]] | [[Category: COT]] | ||
[[Category: alternate conformations]] | [[Category: alternate conformations]] | ||
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[[Category: enzyme-inhibitor complex]] | [[Category: enzyme-inhibitor complex]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:40:00 2008'' |
Revision as of 11:40, 21 February 2008
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Investigation of the Roles of Catalytic Residues in Serotonin N-Acetyltransferase
Overview
Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm. It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling. In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies. Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor. Furthermore, His-120 and His-122 appear to contribute redundantly to this function. By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5. Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom. The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex. Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT. These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors.
About this Structure
1L0C is a Single protein structure of sequence from Ovis aries with as ligand. Active as Aralkylamine N-acetyltransferase, with EC number 2.3.1.87 Full crystallographic information is available from OCA.
Reference
Investigation of the roles of catalytic residues in serotonin N-acetyltransferase., Scheibner KA, De Angelis J, Burley SK, Cole PA, J Biol Chem. 2002 May 17;277(20):18118-26. Epub 2002 Mar 7. PMID:11884405
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