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 asparate 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 D222, H143, T139, H189, and structural waters, is located at the edge of PLP opposite to 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 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 D222, which is directly coupled to the pyridinyl nitrogen, increases the pKa of the pyridine nitrogen and stabilizes the pyridinium cation. H143 and H189 also increase the pKa of the pyridine nitrogen, but, more significantly, influence the position of the proton that resides between D222 and the pyridinyl nitrogen. These findings indicate that the second shell residues directly enhance the rate of catalysis in AAT.
