title: Allosteric reversion of Haemophilus influenzae β-carbonic anhydrase via a proline shift.
abstract: Haemophilus influenzae β-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 β-carbonic anhydrase (PSCA, PDB 1EKJ ), to potentially revert HICA to a nonallosteric enzyme. The W39V/G41A and P48S/A49P mutations decreased the apparent kcat/Km proton dependence from 4 to 2 and 1, respectively, increasing the overall maximal kcat/Km to 16 ± 2 μM(-1) s(-1) (380% of wild type) and 17 ± 3 μM(-1) s(-1) (405% of wild type). The pKa values of the metal-bound water molecule based on the pH-rate profile kinetics (8.32 ± 0.04 for W39V/G41A and 8.3 ± 0.1 for P48S/A49P) were also slightly higher than that for the wild-type enzyme (7.74 ± 0.04). The P48S/A49P variant has lost all pH-rate cooperativity. The W39V/G41A/P48S/A49P variant's kinetics were unusual and were fit with a log-linear function with a slope 0.9 ± 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 β-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-rate 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.
authors.affiliation: Department of Chemistry, Gonzaga University , 502 East Boone Avenue, Spokane, Washington 99258, United States.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Hoffmann
authors.forename: H Rachael
authors.initials: HR
authors.lastname: Million-Perez
authors.forename: Richard
authors.initials: R
authors.lastname: Merkhofer
authors.forename: Hilary
authors.initials: H
authors.lastname: Nicholson
authors.forename: Roger S
authors.initials: RS
authors.lastname: Rowlett
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics*; D018360:Crystallography, X-Ray; D006193:Haemophilus influenzae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D018532:Pisum sativum / Q000737:chemistry / Q000201:enzymology; D017354:Point Mutation; D011392:Proline / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation
chemical_list: D011392:Proline; D002256:Carbonic Anhydrases