Active-site waters

Fig. 11. Necessary elements of the PLC/k mechanism. An active site water is activated for attack on the phosphodiester, a proton is transferred to the departing DAG, and the products leave the active site...

One of the questions that is commonly addressed in mechanistic proposals is how is the active site water activated for nucleophilic attack on the phosphodi-ester bond Numerous combinations of amino acid side chains and zinc ions have been proposed for this role, but there has been little consensus. Critical to all the general base hypotheses is a quite reasonable assumption about catalysis by PLC5c The nucleophilic attack on the phosphodiester moiety proceeds via an in-line mechanism resulting in stereochemical inversion of configuration at phosphorus [86]. While this assumption is consistent with the position of the active site water molecules in the PLCBc-phosphonate inhibitor complex [45], it has not yet been established experimentally. This structure provides a detailed picture of how the amino acid side chains of Glul46, Glu4, Asp55, and the zinc ions interact with the phosphonate inhibitor (Fig. 12), so mechanistic hypotheses now have a structural basis. 

Fig. 12. Distances (A) of PLCBc active site waters from selected amino acid side chains and a phosphonate substrate analogue [45]...

The nature of the reaction catalyzed by PLC i( in which phosphatidylcholine is split into diacylglycerol and phosphorylcholine (Fig. 11) requires two proton transfer steps The first is the deprotonation of an active site water to generate the attacking hydroxide nucleophile, and the second is the protonation of the alkoxide leaving group. Although analyses of the X-ray structures of PLCSc and... 

CA has been the snbject of numerous theoretical studies, most of which have centered on differentiating between the Lipscomb and Lindskog mechanisms or on the proton-transfer step. Early studies concentrated on substrate binding and simple models for the active site jqqj, mechanistic studies on model active sites were being published229.254- 268 suggested quite early that active-site water molecules may... 

The importance of maintaining the active site water network in CA II for efficient proton transfer was investigated by substituting different amino acids of varying size at position 65 and measuring the rate constants for proton transfer in the variant carbonic anhydrases... 

There is general agreement that carbonic anhydrase activity is linked to the zinc ion and its ligands. At the active site water is bound to the zinc but the state and involvement of the water in the actual catalysis is a matter of controversy. According to a widely accepted opinion the zinc-bound water is ionized and the activity is a function of pH1S6. ... 

C. Heiss, M. Laivenieks, G. J. Zeikus, and R. S. Phillips, The stereospecificity of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus is partially determined by active site water,... 

Including Active Site Water Molecules An Example... 

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