Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase.
The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.
Smith
K S
KS
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Cosper
N J
NJ
Stalhandske
C
C
Scott
R A
RA
Ferry
J G
JG
D015502:Absorptiometry, Photon / Q000379:methods; D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D007700:Kinetics; D017014:Methanobacterium / Q000201:enzymology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation / Q000379:methods
D002256:Carbonic Anhydrases