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PDBsum entry 5tot
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References listed in PDB file
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Key reference
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Title
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Direct evidence that an extended hydrogen-Bonding network influences activation of pyridoxal 5'-Phosphate in aspartate aminotransferase.
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Authors
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S.Dajnowicz,
J.M.Parks,
X.Hu,
K.Gesler,
A.Y.Kovalevsky,
T.C.Mueser.
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Ref.
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J Biol Chem, 2017,
292,
5970-5980.
[DOI no: ]
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PubMed id
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Abstract
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Pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor
used to catalyze a wide variety of chemical reactions involved in amino acid
metabolism. PLP-dependent enzymes optimize specific chemical reactions by
modulating the electronic states of PLP through distinct active site
environments. In aspartate aminotransferase (AAT), an extended hydrogen bond
network is coupled to the pyridinyl nitrogen of the PLP, influencing the
electrophilicity of the cofactor. This network, which involves residues Asp-222,
His-143, Thr-139, His-189, and structural waters, is located at the edge of PLP
opposite the reactive Schiff base. We demonstrate that this hydrogen bond
network directly influences the protonation state of the pyridine nitrogen of
PLP, which affects the rates of catalysis. We analyzed perturbations caused by
single- and double-mutant variants using steady-state kinetics, high resolution
X-ray crystallography, and quantum chemical calculations. Protonation of the
pyridinyl nitrogen to form a pyridinium cation induces electronic delocalization
in the PLP, which correlates with the enhancement in catalytic rate in AAT.
Thus, PLP activation is controlled by the proximity of the pyridinyl nitrogen to
the hydrogen bond microenvironment. Quantum chemical calculations indicate that
Asp-222, which is directly coupled to the pyridinyl nitrogen, increases the pKa
of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and
His-189 also increase the pKa of the pyridine nitrogen but, more significantly,
influence the position of the proton that resides between Asp-222 and the
pyridinyl nitrogen. These findings indicate that the second shell residues
directly enhance the rate of catalysis in AAT.
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