The Mechanism of Action of Carbonic Anhydrase and LpxC
EMSL Project ID
17794
Abstract
Zinc binding sites in proteins play diverse roles, including enhancement or inhibition of catalysis, stabilization and assembly of protein structures, and regulation of metal concentration. Further development of potent and specific inhibitors of metalloenzymes will be aided by additional knowledge of the variations in catalytic mechanisms and molecular recognition properties of these enzymes, including elucidation of the role(s) of the catalytic zinc ions. Many advantageous properties of Zn2+ contribute to its use as a cofactor in catalysis. Foremost, zinc is a strong Lewis acid that is not redox reactive, since it has a full shell of d electrons (d10 configuration). Zinc ions catalyze reactions by coordinating a nucleophilic water to lower its pKa; coordinating and polarizing a substrate molecule (such as a carbonyl oxygen or a thiolate); and/or stabilizing developing negative charge in a transition state. Secondly, Zn2+ is able to adopt a range of coordination numbers (4-6) and geometric configurations that allow the metal to accommodate structural rearrangements that can occur during the course of chemical reactions. Finally, although zinc is generally tightly bound to proteins, it can undergo rapid ligand exchange necessary for efficient catalysis. A comprehensive understanding about the catalytic mechanisms of zinc metalloenzymes, as well as the development of metalloenzyme inhibitors, will be facilitated by knowledge about the metal-water pKa, understanding the electrostatic environment around the metal site and a further understanding of active site proton relay systems. Traditionally the pKa value of the metal-water has been inferred either by using metal-substitution in combination with kinetic experiments and/or cobalt absorption spectroscopy. Examination of the pH-dependence of the reaction of interest under kcat/KM or kcat conditions may or may not provide information about the metal-water ionization depending on conditions used for experiments. A shift in an observed pKa value upon substitution with different metal ions indicates a metal dependent ionization. However, the accurate determination of the intrinsic pKa value(s) from these data is complicated by either kinetic perturbations that arise from commitment factors and/or because of difficulties in assigning pKa values from bell-shaped curves, as is often observed for zinc metalloenzymes. In the latter approach, the pH-dependence of the d-d transitions in the absorption spectrum of Co(II)-substituted enzymes is determined and reflects ionization of Co(II)-H2O, as previously observed for both carboxypeptidase A and carbonic anhydrase. This technique has been used successfully to measure the pKa of metal-bound water molecules in other metalloenzymes; however this method only provides direct information about the ionization of a non-native metal ion. The direct determination of the zinc-water ionization using 67Zn NMR circumvents the problems that are associated with the methods described above. This technique provides a direct measurement of the intrinsic pKa value of the metal of interest, and does not suffer from the complications that arise from kinetic perturbations or metal substitution.
Project Details
Project type
Capability Research
Start Date
2006-04-06
End Date
2007-03-20
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Lipton AS, RW Heck, M Hernick, CA Fierke, and PD Ellis. 2008. "Residue Ionization in LpxC Directly Observed by 67Zn NMR Spectroscopy." Journal of the American Chemical Society 130(38):12671-12679.