4WAK

H. influenzae beta-carbonic anhydrase variant W39V/G41A

Summary for 4WAK

• Entry DOI: 10.2210/pdb4wak/pdb
• Related: 2A8D 4WAJ 4WAM
• Descriptor: Carbonic anhydrase 2, ZINC ION, BICARBONATE ION, ... (5 entities in total)
• Functional Keywords: carbonic anhydrase allosteric site, lyase
• Biological source: Haemophilus influenzae
• Total number of polymer chains: 2
• Total formula weight: 52719.81

Authors

Hoffmann, K.M.,Rowlett, R.S. (deposition date: 2014-08-29, release date: 2014-12-31, Last modification date: 2023-09-27)

Primary citation

Hoffmann, K.M.,Million-Perez, H.R.,Merkhofer, R.,Nicholson, H.,Rowlett, R.S.
Allosteric Reversion of Haemophilus influenzae beta-Carbonic Anhydrase via a Proline Shift.
Biochemistry, 54:598-611, 2015

PubMed 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.

PubMed: 25506786
DOI: 10.1021/bi501116e

Experimental method

X-RAY DIFFRACTION (2.49 Å)