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

Abstract

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 stabilizesmore » 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.« less

Authors:
 [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
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1352796
Grant/Contract Number:  
AC05-00OR22725; AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 292; Journal Issue: 14; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; enzyme catalysis; hydrogen bond; pyridoxal phosphate; quantum chemistry; x-ray crystallography

Citation Formats

Dajnowicz, Steven, Parks, Jerry M., Hu, Xiche, Gesler, Korie, Kovalevsky, Andrey Y., and Mueser, Timothy C. Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase. United States: N. p., 2017. Web. doi:10.1074/jbc.M116.774588.
Dajnowicz, Steven, Parks, Jerry M., Hu, Xiche, Gesler, Korie, Kovalevsky, Andrey Y., & Mueser, Timothy C. Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase. United States. doi:10.1074/jbc.M116.774588.
Dajnowicz, Steven, Parks, Jerry M., Hu, Xiche, Gesler, Korie, Kovalevsky, Andrey Y., and Mueser, Timothy C. Thu . "Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase". United States. doi:10.1074/jbc.M116.774588. https://www.osti.gov/servlets/purl/1352796.
@article{osti_1352796,
title = {Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase},
author = {Dajnowicz, Steven and Parks, Jerry M. and Hu, Xiche and Gesler, Korie and Kovalevsky, Andrey Y. and Mueser, Timothy C.},
abstractNote = {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.},
doi = {10.1074/jbc.M116.774588},
journal = {Journal of Biological Chemistry},
number = 14,
volume = 292,
place = {United States},
year = {Thu Feb 23 00:00:00 EST 2017},
month = {Thu Feb 23 00:00:00 EST 2017}
}

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