His-163 is a stereospecific proton donor in the mechanism of d-glucosaminate-6-phosphate ammonia-lyase

Abstract

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) catalyzes the conversion of d-glucosaminate-6-phosphate to 2-keto-3-deoxyglutarate-6-phosphate, with stereospecific protonation of C-3 of the product. The crystal structure of DGL showed that His-163 could serve as the proton donor. H163A mutant DGL is fully active in the steady-state reaction, and the pre-steady-state kinetics are very similar to those of wild-type DGL. However, H163A DGL accumulates a transient intermediate with λ_max at 293 nm during the reaction that is not seen with wild-type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D₂ O shows that the product is a mixture of deuterated diastereomers at C-3. These results establish that His-163 is the proton donor in the reaction mechanism of DGL.

Keywords: aminoacrylate intermediate; elimination reaction; pyridoxal-5′-phosphate; stereochemistry.

Fig. 1

Crossed‐eye stereo view of the structure of DGL (PDB 7CLE), showing the proximity of His‐163 to the PLP. This figure was prepared with Pymol 2.3.0 (PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC, New York, NY, USA).

Fig. 2

Rapid‐scanning stopped‐flow spectroscopy of H163A and wild‐type DGL with DGA‐6‐P in the early part of the reaction. (A) Reaction of H163A DGL. The enzyme spectrum is shown with the dashed magenta line. The first spectrum after mixing (0.001 s) is shown in black. The second spectrum is at 0.04 s (blue). The third spectrum is at 1.2 s (red). (B) Time courses for the reaction of H163A DGL in the early part of the reaction. Black, 489 nm; red, 422 nm; blue, 293 nm. (C) Reaction of wild‐type DGL. The enzyme spectrum is shown with the dashed magenta line. The first spectrum after mixing (0.001 s) is shown in black. The second spectrum is at 0.04 s (blue). The third spectrum is at 0.9 s (red). (D) Time courses for the reaction of wild‐type DGL in the early part of the reaction. Black, 488 nm; red, 422 nm; blue, 294 nm.

Fig. 3

Time courses and SVD spectra for the reaction of H163A and wild‐type DGL with DGA‐6‐P. (A) Time courses for H163A DGL. Black, 293 nm; red, 322 nm; blue, 421 nm. (B) Time courses for wild‐type DGL. Black, 293 nm; red, 322 nm; blue, 421 nm. (C) SVD spectra for reaction of H163A DGL. Black, initial spectrum; red, intermediate spectrum; blue, final spectrum. The inset shows the difference between the black and red SVD spectra. (D) SVD spectra for reaction of wild‐type DGL. Black, initial spectrum; red, intermediate spectrum; blue, final spectrum.

Fig. 4

¹H‐NMR spectra of KDG‐6‐P from reaction of wild‐type and H163A DGL. Black, spectrum in D₂O of KDG‐6‐P from reaction of wild‐type DGL with DGA‐6‐P in H₂O. Red, spectrum of KDG‐6‐P from reaction of wild‐type DGL with DGA‐6‐P in D₂O. Blue, spectrum of KDG‐6‐P from reaction of H163A DGL with DGA‐6‐P in D₂O. The two sets of peaks arise from the α‐ and β‐anomers.

Scheme 1

Mechanism of H163A DGL.

Fig. 5

Crossed‐eye stereo structure of DGA‐6‐P docked manually into the active site of DGL (PDB 7LCE) to give a gem‐diamine.