PNNL

Abstract

The pH dependence of the 67Zn NMR spectroscopy has been measured for both wild type (WT) and the H265A mutant of LpxC, each in the resting state – absence of substrate. We find for the WT that the pH 6 NMR spectrum contains two overlapping quadrupole lineshapes with Cq’s of 10 and 12.9 MHz. The spectrum measured on the sample prepared at a pH near 9 is dominated by the appearance of a third species with a Cq of 14.3 MHz. This is consistent with the two pKa’s previously inferred by the bell-shaped dependence of the LpxC catalyzed reaction. Comparing the experimental results with predictions from QM/MM modeling we suggest that pKa1 (low pH) represents the ionization of Glu78 and pKa2 (high pH) reflects the ionization of His265. These results are consistent with water being bound to the Zn2+ thoughout this pH range. The 67Zn NMR spectra of the mutant H265A seem invariant to pH with a Cq of 9.55 MHz sufficient to describe both low and high pH data. The QM/MM models of H265A suggest that water is bound to the zinc and Glu78 is protonated over this pH range.