Residue lonization in LpxC Directly Observed by ~(67)Zn NMR Spectroscopy

摘要

The pH dependence of the solid-state ~(67)Zn NMR lineshapes has been measured for both the wild type (WT) and the H265A mutant of Aquifex aeolicus LpxC, each in the absence of substrate (resting state). The ~(67)Zn NMR spectrum of WT LpxC at pH 6 (prepared at 0 ℃) contains two overlapping quadrupole lineshapes with C_q values of 10 and 12.9 MHz, while the spectrum measured for the sample prepared at a pH near 9 (at 0 ℃) is dominated by the appearance of a third species with a C_q of 14.3 MHz. These findings are consistent with the two pK_a values previously observed by the bell-shaped dependence of the LpxC-catalyzed reaction. On the basis of comparison of the experimental results with predictions from quantum mechanical/molecular mechanical (QM/MM) modeling, we suggest that pK_(a1) (low pH) represents the ionization of Glu78 and pK_(a2) (high pH) reflects the ionization of another active site residue located near the zinc ion, such as His265. These results are also consistent with water being bound to the Zn~(2+) ion throughout this pH range. The ~(67)Zn NMR spectra of the H265A mutant appear to be pH independent, with a C_q of 9.55 MHz being sufficient to describe both low- and high-pH data. The QM/MM models of the H265A mutant suggest that over this pH range water is bound to the zinc ion while Glu78 is protonated.