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

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 pK_a of the pyridine nitrogen and stabilizes the pyridinium cation. His-143 and His-189 also increase the pK_a 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.

Keywords: X-ray crystallography; enzyme catalysis; hydrogen bond; pyridoxal phosphate; quantum chemistry.

Figure 1.

A, chemical structures and atom labels for pyridoxal 5′-phosphate, internal aldimine, external aldimine, and pyridoxamine 5-phosphate. B, overall fold of the biological and crystallographic homodimer of AAT. Stars indicate the unresolved active site cap in chain B. C, two half-reactions that occur in the ping-pong bi-bi mechanism for AAT.

Figure 2.

Simplified mechanism for the first half-reaction of AAT.

Figure 3.

Active site of AAT chain A.

Figure 4.

Atomic coordinates of active site residues in the microenvironment near PLP-N1. A, AAT_WT chain A; B, AAT_WT chain B; C, D222T chain A; D, D222T chain B; E, H143L chain A; F, H143L chain B; G, H143L/H189L chain A; H, H143L/H189L chain B. C, E, and G, the black carbon scheme shows resolved alternate conformations of the active site residues in each respective X-ray structure. The blue mesh is the omitted |F_o − F_c| electron density contoured at 3σ for the newly observed water molecules in D and E.

Figure 5.

Structure of wild-type AAT color-coded by overall B-factor. Cool colors (blue) represent low B-factors, and warm colors (yellow and orange) represent high B-factors. Asterisks indicate the unresolved active site cap in chain B.

Scheme 1.

Figure 6.

A, optimized geometry of the AAT_WT model. The PLP-N1 proton is shown in green. Some hydrogens are omitted for clarity. B, relaxed potential energy scan for AAT_WT.

Figure 7.

A, C, and E show the optimized geometries for the D222T, H143L, and H143L/H189L QM models. The optimized geometries for structures I, II, and III are the reactant, TS, and product, respectively, for each model. Only PLP, four active site residues, and one water molecule are shown for simplicity. The green atoms are the protons of interest. B, D, and F show the relative electronic energy (solid black line) for D222T, H143L, and H143L/H189L, respectively. The dashed red lines are the enthalpies relative to the TS (structure II).