Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active site variant (D44N) that is incapable of forming a strong zinc ligand. The mutation of Trp39 to Phe increases the apparent Ki for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active site conformation nearly identical to that of non-allosteric beta-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.
Allosteric Site Variants of Haemophilus influenzae beta-Carbonic Anhydrase.,Rowlett RS, Tu C, Lee J, Herman AG, Chapnick DA, Shah SH, Gareiss PC Biochemistry. 2009 May 21. PMID:19459702
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↑ Rowlett RS, Tu C, Lee J, Herman AG, Chapnick DA, Shah SH, Gareiss PC. Allosteric Site Variants of Haemophilus influenzae beta-Carbonic Anhydrase. Biochemistry. 2009 May 21. PMID:19459702 doi:10.1021/bi900663h