Kinetic and Spectroscopic Characterization of the Gamma-Carbonic Anhydrase from the Methanoarchaeon Methanosarcina thermophila

Abstract

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.

A Role for Iron in an Ancient Carbonic Anhydrase

“…Kinetic investigations of the ␣ class CAs reveal a "zinc hydroxide" mechanism for catalysis (8) that also extends to both the ␤ and ␥ classes (9,10). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps,…”

“…All of the biochemically characterized CAs from the ␣, ␤, and ␥ classes are categorically described as zinc metalloenzymes; however, there are few reports investigating the role of metals other than zinc. Although in vitro replacement of zinc with cobalt in a few purified CAs from the ␣ and ␥ classes yields robustly active enzymes (10,13,14), it is unknown whether any cobalt-containing CAs are synthesized in vivo. A CA from the marine diatom Thalassiosira weissflogii contains cadmium (15), although there are no reports of a biochemical or structural characterization of the purified enzyme.…”

“…Carbon dioxide hydrating activity was measured by stopped flow spectroscopy using the changing pH indicator method as described previously (10), except for enzymes purified anaerobically, in which case all of the solutions were anaerobic. The protein concentration determinations were performed as described (10). The Mössbauer spectra were recorded on a spectrometer (WEB Research, Edina, MN) operating in the constant acceleration mode in a transmission geometry.…”

Structural and Kinetic Characterization of an Archaeal β-Class Carbonic Anhydrase

“…The kinetic properties reported for the P. sativum and Spinacia oleracea (spinach) ␤-class carbonic anhydrases are also consistent with this mechanism (29,30,46). Thus, the kinetic analyses of enzymes from all three classes suggest convergent evolution of the catalytic mechanism (1,36,39). The ␤-class was initially thought to be composed solely of carbonic anhydrases from monocotyledenous and dicotyledenous plants.…”

“…2D). The active site zinc of the ␣-and ␥-class enzymes is coordinated by three histidines and at least one water molecule (1,28,61). In the catalytic mechanism of carbonic anhydrase, the zinc-bound water ionizes to a metal-bound hydroxide ion that attacks CO 2 (equation 2a).…”