Affiliation:
1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802,1 and
2. Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602-25562
Abstract
ABSTRACT
The β-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) β-class carbonic anhydrase indicate that the secondary structure of the β-class enzymes is predominantly α-helical, unlike that of the α- or γ-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 β-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic β-class enzymes and firmly establish that the α-, β-, and γ-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
46 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献