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

Abstract

Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize 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 CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which 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-nitrophenylacetate 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 replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) 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 CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment 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.

Figure 1

Western blot analysis of membrane-associated and soluble forms of human CA XII. Cell extracts expressing wild-type CA XII or purified soluble Q291X CA XII and H64A and H64R derivatives of Q291X CA XII were analyzed by SDS/PAGE followed by Western blot using a polyclonal antibody against human CA XII. The apparent molecular masses of the major polypeptides are indicated in kDa.

Figure 2

The kinetic constant k_cat/K_m (M⁻¹⋅s⁻¹) for the hydration of CO₂ catalyzed by (wild-type) human CA XII. Data were measured by stopped-flow spectrophotometry at 25°C using 25 mM of the buffers listed in the text. The total ionic strength was maintained at 0.2 M by addition of Na₂SO₄. Solutions also contained 5 μM EDTA. The solid line is a least-squares fit to a single ionization with pK_a = 7.1 ± 0.1 and with a maximum of (3.4 ± 0.3) × 10⁷ M⁻¹⋅s⁻¹.

Figure 3

The kinetic constant k_cat/K_m (M⁻¹⋅s⁻¹) for the hydrolysis of 4-nitrophenylacetate catalyzed by human CA XII. Measurements were made at 25°C using solutions maintained at an ionic strength of 0.2 M using Na₂SO₄ and containing 5 μM EDTA and 25 mM of one of the buffers listed in the text. The solid line is a least-squares fit with the maximal of k_cat/K_m at 90 ± 2 and pK_a 7.1 ± 0.1.

Figure 4

The pH dependence of the steady-state turnover number k_cat (s⁻¹) for the hydration of CO₂ catalyzed by human CA XII determined by stopped-flow spectrophotometry at 25°C. Solutions contained 5 μM EDTA and 25 mM of one of the buffers listed in Materials and Methods. The solid line is a least-squares fit with a maximal value of k_cat at (4.0 ± 1.7) × 10⁵ s⁻¹ and an apparent pK_a of 8.0 ± 0.2.

Figure 5

The pH dependence of R_H2O/[E] (s⁻¹) catalyzed by human wild-type CA XII (●), H64A CA XII (▵), and H64R CA XII (■). Conditions were as described in Fig. 2. The solid line for wild-type CA XII is a least-squares fit of Eq. 4 to the data yielding (pK_a)_acceptor = 6.1 ± 0.1; (pK_a)_donor = 7.5 ± 0.1; and k_B = (2.9 ± 0.2) × 10⁵ s⁻¹.

Figure 6

The pH dependence of R_H2O/[E] (s⁻¹) catalyzed by human CA XII (●) and membrane-bound CA XII (□). Conditions were as described in Fig. 2.