Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers

Citation
B. Ulmasov et al., Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers, P NAS US, 97(26), 2000, pp. 14212-14217
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
0027-8424 → ACNP
Volume
97
Issue
26
Year of publication
2000
Pages
14212 - 14217
Database
ISI
SICI code
0027-8424(200012)97:26<14212:PAKAOR>2.0.ZU;2-V
Abstract
Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an act ive extracellular CA domain that is overexpressed on cell surfaces of certa in cancers. Its expression has been linked to tumor invasiveness. To charac terize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO2 were measured at steady state by stopped-flow spectro photometry and at chemical equilibrium by the exchange of O-18 between CO2 and water determined by mass spectrometry. The catalysis of CO2 hydration b y soluble CA XII has a maximal value of k(cat)/K-cm at 34 muM(-1).s(-1), wh ich is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitr ophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the re placements His64 --> Ala and His64 --> Arg, as well as by the selective inh ibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO2 hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO2/HCO3- homeostasis in cells in which it is normally expres sed. They are also compatible with a role for CA XII in acidifying the micr oenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.