Crystallography and Its Impact on Carbonic Anhydrase Research

Abstract

X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO₂, producing HCO₃ ^- and a proton. The CA structure and ping-pong mechanism have been extensively studied and are well understood. Though the function of CA plays an important role in a variety of physiological functions, CA has also been associated with diseases such as glaucoma, edema, epilepsy, obesity, and cancer and is therefore recognized as a drug target. In this review, a brief history of crystallography and its impact on CA research is discussed.

Figure 1

CA protein database entries growth of CA PDB entries since 1980. Blue represents all the past years entries whereas orange represents entries submitted that year.

Figure 2

Human CA isoform structures: (a) CA I, (b) CA II, (c) CA III, (d) CA IV, (e) CAVI, (f) CA VII, (g) CA VIII, (h) CA IX, (i) CA XII, (j) CA XIII, and (k) CA XIV.

Figure 3

CA families structural examples of CAs from (a)α (gray), (b) β (red), (c) γ (blue), and (d)ζ (green) families.

Figure 4

Active site mechanism. The surface rendition of CA II with zinc bound to active site residues H94, H96, and H119. The hydrophobic side is depicted in orange while the hydrophilic side is depicted in purple. Blue arrows depict substrate flow, CO₂, and HCO₃⁻ through the hydrophobic side and proton transfer through the hydrophilic side. A cartoon depiction of α CA ping-pong mechanism.

Figure 5

CA inhibitors structural formulae of clinically relevant CAIs.

Figure 6

Pockets in α CAs. (a) Hydrophilic and hydrophobic regions of the active site have been colored purple and orange, respectively. The hydrophobic pockets (b) 1 and (c) 2 have been shaded yellow and green, respectively. (d) Isoform unique residues that constitute the selective pocket are shown in blue (Table 2).

Figure 7

Selective zones for human α CAs The three colored zones represent isoform unique residues in human CAs. Zones are located 5-10 (pink), 10-15 (yellow), and 15-20 (blue) Å from the catalytic zinc (Table 2).