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PDBsum entry 1l0c
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Investigation of the roles of catalytic residues in serotonin n-Acetyltransferase.
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Authors
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K.A.Scheibner,
J.De angelis,
S.K.Burley,
P.A.Cole.
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Ref.
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J Biol Chem, 2002,
277,
18118-18126.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
Fig. 1. Mechanism of the acetyltransferase reaction
catalyzed by AANAT (A) and alkyl transfer between CoASH and
N-bromoacetyltryptamine (compound 1), resulting in the
bisubstrate analog inhibitor (compound 2) (B).
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Figure 2.
Fig. 2. X-ray structure of AANAT bound to the bisubstrate
analog (compound 2). Tyr-168, His-120, and His-122 are
highlighted and were the focus of the mutational studies
detailed in this report.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2002,
277,
18118-18126)
copyright 2002.
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