Title: Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [3];  [3];  [4];  [3]
  1. Univ. of Toledo, OH (United States). DEpt. of Chemistry and Biochemistry; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biology and Soft Matter Division
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). University of Tennessee and Oak Ridge National Lab. Center for Molecular Biophysics
  3. Univ. of Toledo, OH (United States). DEpt. of Chemistry and Biochemistry
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biology and Soft Matter Division
+ Show Author Affiliations

We used pyridoxal 5'-phosphate (PLP) is a fundamental, multifunctional enzyme cofactor 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. Therefore, 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. Our findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.

Research Organization:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC02-06CH11357; AC05-00OR22725
OSTI ID:
1352796
Journal Information:
Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 14 Vol. 292; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (6)

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Low‐Barrier and Canonical Hydrogen Bonds Modulate Activity and Specificity of a Catalytic Triad journal September 2019
Low‐Barrier and Canonical Hydrogen Bonds Modulate Activity and Specificity of a Catalytic Triad journal November 2019
Investigations into the flexibility of the 3D structure and rigid backbone of quinoline by fluorine addition to enhance its blue emission journal January 2018
Hyperconjugation promoted by hydrogen bonding between His98/His241 and a carboxyl group contributes to tyrosine decarboxylase catalysis journal January 2019
Dimerization misalignment in human glutamate-oxaloacetate transaminase variants is the primary factor for PLP release journal September 2018

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
enzyme catalysis
hydrogen bond
pyridoxal phosphate
quantum chemistry
x-ray crystallography