The catalytic mechanism of carbonic anhydrase includes the reaction of a zinc-bound hydroxide ion with the CO, substrate. This hydroxide ion is part of a hydrogen-bonded network involving the conserved amino acid residues Thr199, Glu106 and Tyr7. To investigate the functional importance of these residues, a number of site-specific mutants have been made. Thus, Thr199 has been changed to Ala, Glu106 to Ala, Gln and Asp, and Tyr7 to Phe. The effects of these mutations on catalyzed CO, hydration and ester hydrolysis have been measured, as well as the binding of some inhibitors. The results show that the CO, hydration activity of the mutant with Phe7 is only marginally reduced, whereas the esterase activity is larger than that of unmodified enzyme. It is concluded that Tyr7 is not a functionally required element of the hydrogen-bonded network. The CO, hydration activity (kcat as well as kCJKm) and the esterase activity of the mutant with Ala199 are reduced about 100-fold. The affinity for the sulfonamide inhibitor, dansylamide, is only slightly reduced while the mutant has an enhanced affinity for bicarbonate and the anionic inhibitor, SCN-. The activities of the mutants with Ala106 and Gln106 are also reduced. The reduction of the esterase activity is about 100-fold, while k,,, for CO, hydration has decreased by a factor of 1OOO. The parameter kcat/ K , is only about one order of magnitude smaller for these mutants than for the unmodified enzyme. The binding of dansylamide and another sulfonamide inhibitor, acetazolamide, are about 20-times weaker to the mutant with Gln106 than to unmodified enzyme. These results suggest important roles for Thr199 and Glu106 in carbonic anhydrase catalysis. The mutant with Asp106 is almost fully active suggesting that the structure has undergone a compensatory change to maintain the interaction between residue 106 and Thr199.It has long been known that the zinc ion in the active site of carbonic anhydrase is required for catalysis of the reversible hydration of CO, [l, 21 as well as for catalysis of ester hydrolysis [2, 31. In addition to the three histidine residues, 94, 96 and 119, which chelate the zinc ion in a very stable complex, the active site contains a number of conserved amino acid residues, which are connected to the metal-ion center through networks of hydrogen bonds [4]. One of these networks is shown schematically in Fig.

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To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a 'proton shuttle group' during catalysis of CO,-HCOs-interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site-directed mutagenesis. All these variants turned out to have high CO, hydration activities. The k,, values at pH 8.8and 25°C were only reduced by 1.53.5fold compared to the unmodified enzyme. These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction. The variants also catalyze the hydrolysis of 4-nitrophenyl acetate. The pK, values for the activity-controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major pK, shifted to 8.4

Novel accessible scaffold featuring circularly‐polarized room‐temperature phosphorescence (RTP) is attractive but challenging. Herein, we report a new feasibly separable bidibenzo[b,d]furan compound with strong circularly‐polarized RTP when doping into the rigid polymer matrix. The simple silica‐gel column can separate the absolute chiral R‐isomer with excellent chiroptical properties. The experimental data reveal that the treated films exhibit an RTP efficiency of 14.8 %, a largest dissymmetric factor of 0.12, and a longest lifetime of 0.56 s under ambient conditions. It is found that reducing the nonradiative decays boosts the intrinsic circularly‐polarized RTP emission. The impressive results indicate that the locked axial chirality skeleton endows the potential of achieving superior circularly‐polarized emission for the small organic optoelectronic molecules.

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