Allosteric Reversion of Haemophilus influenzae 尾-Carbonic Anhydrase via a Proline Shift

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• 作者：Katherine M. Hoffmann ; H. Rachael Million-Perez ; Richard Merkhofer ; Hilary Nicholson ; Roger S. Rowlett
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：January 20, 2015
• 年：2015
• 卷：54
• 期：2
• 页码：598-611
• 全文大小：665K
• ISSN：1520-4995

文摘

Haemophilus influenzae 尾-carbonic anhydrase (HICA) has been reverse-engineered in the allosteric site region to resemble the nonallosteric Pisum sativum enzyme in order to identify critical features of allostery and intersusbunit communication. Three variants (W39V/G41A, P48S/A49P, and W39V/G41A/P48S/A49P) were identified, through a comparison with a crystal structure of nonallosteric P. sativum 尾-carbonic anhydrase (PSCA, PDB 1EKJ), to potentially revert HICA to a nonallosteric enzyme. The W39V/G41A and P48S/A49P mutations decreased the apparent k_cat/K_m proton dependence from 4 to 2 and 1, respectively, increasing the overall maximal k_cat/K_m to 16 卤 2 渭M^鈥? s^鈥? (380% of wild type) and 17 卤 3 渭M^鈥? s^鈥? (405% of wild type). The pK_a values of the metal-bound water molecule based on the pH鈥搑ate profile kinetics (8.32 卤 0.04 for W39V/G41A and 8.3 卤 0.1 for P48S/A49P) were also slightly higher than that for the wild-type enzyme (7.74 卤 0.04). The P48S/A49P variant has lost all pH鈥搑ate cooperativity. The W39V/G41A/P48S/A49P variant鈥檚 kinetics were unusual and were fit with a log鈥搇inear function with a slope 0.9 卤 0.2. The crystal structure of the W39V/G41A variant revealed an active site very similar to the T-state wild-type oligomer with bicarbonate trapped in the escort site. By contrast, the X-ray crystal structure of a proline shift variant (P48S/A49P) reveals that it has adopted an active site conformation nearly identical to that of nonallosteric 尾-carbonic anhydrase (R-state) for one chain, including a tight association with the dimer-exchanged N-terminal helices; the second chain in the asymmetric unit is associated in a biologically relevant oligomer, but it adopts a T-state conformation that is not capped by dimer-exchanged N-terminal helices. The hybrid R/T nature of HICA P48S/A49P structurally recapitulates the interruption of pH鈥搑ate cooperativity observed for this variant. Comparison of the conformations of the R and T chains of P48S/A49P suggests a new hypothesis to explain HICA allosteric communication that is mediated by the N-terminal helices and anion binding at the dimer interface.