Kinetic and crystallographic studies of the role of tyrosine 7 in the active site of human carbonic anhydrase II.

The rate limiting step in catalysis of bicarbonate dehydration by human carbonic anhydrase II (HCA II) is an intramolecular proton transfer from His64 to the zinc-bound hydroxide. We have examined the role of Tyr7 using site-specific mutagenesis and measuring catalysis by the ¹8O exchange method using membrane inlet mass spectrometry. The side chain of Tyr7 in HCA II extends into the active-site cavity about 7 Å from the catalytic zinc atom. Replacement of Tyr7 with eight other amino acids had no effect on the interconversion of bicarbonate and CO2, but in some cases caused enhancements in the rate constant of proton transfer by nearly 10-fold. The variant Y7I HCA II enhanced intramolecular proton transfer approximately twofold; its structure was determined by X-ray crystallography at 1.5 Å resolution. No changes were observed in the ordered solvent structure in the active-site cavity or in the conformation of the side chain of the proton shuttle His64. However, the first 11 residues of the amino-terminal chain in Y7I HCA II assumed an alternate conformation compared with the wild type. Differential scanning calorimetry showed variants at position 7 had a melting temperature approximately 8 °C lower than that of the wild type.

Design of a carbonic anhydrase IX active-site mimic to screen inhibitors for possible anticancer properties.

Recently, a convincing body of evidence has accumulated suggesting that the overexpression of carbonic anhydrase isozyme IX (CA IX) in some cancers contributes to the acidification of the extracellular matrix, which in turn promotes the growth and metastasis of the tumor. These observations have made CA IX an attractive drug target for the selective treatment of certain cancers. Currently, there is no available X-ray crystal structure of CA IX, and this lack of availability has hampered the rational design of selective CA IX inhibitors. In light of these observations and on the basis of structural alignment homology, using the crystal structure of carbonic anhydrase II (CA II) and the sequence of CA IX, a double mutant of CA II with Ala65 replaced by Ser and Asn67 replaced by Gln has been constructed to resemble the active site of CA IX. This CA IX mimic has been characterized kinetically using (18)O-exchange and structurally using X-ray crystallography, alone and in complex with five CA sulfonamide-based inhibitors (acetazolamide, benzolamide, chlorzolamide, ethoxzolamide, and methazolamide), and compared to CA II. This structural information has been evaluated by both inhibition studies and in vitro cytotoxicity assays and shows a correlated structure-activity relationship. Kinetic and structural studies of CA II and CA IX mimic reveal chlorzolamide to be a more potent inhibitor of CA IX, inducing an active-site conformational change upon binding. Additionally, chlorzolamide appears to be cytotoxic to prostate cancer cells. This preliminary study demonstrates that the CA IX mimic may provide a useful model to design more isozyme-specific CA IX inhibitors, which may lead to development of new therapeutic treatments of some cancers.

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1).

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.