5TOT

Crystal structure of AAT H143L:H189L double mutant

Summary for 5TOT

• Entry DOI: 10.2210/pdb5tot/pdb
• Related: 5TON 5TOQ 5TOR
• Descriptor: Aspartate aminotransferase, cytoplasmic (2 entities in total)
• Functional Keywords: transferase
• Biological source: Sus scrofa (Pig)
• Cellular location: Cytoplasm: P00503
• Total number of polymer chains: 2
• Total formula weight: 93519.81

Authors

Mueser, T.C.,Dajnowicz, S.,Kovalevsky, A. (deposition date: 2016-10-18, release date: 2017-03-01, Last modification date: 2023-11-15)

Primary citation

Dajnowicz, S.,Parks, J.M.,Hu, X.,Gesler, K.,Kovalevsky, A.Y.,Mueser, T.C.
Direct evidence that an extended hydrogen-bonding network influences activation of pyridoxal 5'-phosphate in aspartate aminotransferase.
J. Biol. Chem., 292:5970-5980, 2017

PubMed Abstract: 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 p of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the p 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.

PubMed: 28232482
DOI: 10.1074/jbc.M116.774588

Experimental method

X-RAY DIFFRACTION (1.4 Å)