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
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.