Friday, September 19, 2008

J. Am. Chem. Soc., 130 (38), 12671–12679, 2008.

Residue Ionization in LpxC Directly Observed by 67Zn NMR Spectroscopy

Andrew S. Lipton,† Robert W. Heck,† Marcy Hernick,‡§ Carol A. Fierke,*‡ and Paul D. Ellis*†

The pH dependence of the solid-state 67Zn 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 67Zn NMR spectrum of WT LpxC at pH 6 (prepared at 0 °C) contains two overlapping quadrupole lineshapes with Cq values of 10 and 12.9 MHz, while the spectrum measured for the sample prepared at a pH near 9 (at 0 °C) is dominated by the appearance of a third species with a Cq of 14.3 MHz. These findings are consistent with the two pKa 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 pKa1 (low pH) represents the ionization of Glu78 and pKa2 (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 Zn2+ ion throughout this pH range. The 67Zn NMR spectra of the H265A mutant appear to be pH independent, with a Cq 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.

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