Role of Water in Enzyme Active Sites

Interactions between specific solvent molecules and protein atoms are important for protein structure and function. Water molecules participate directly in many enzymatic reactions, including those catalyzed by the large class of enzymes that hydrolyze peptide bonds. X-ray crystallography has identified waters that appear to be an integral part of the structure of a protein. Some of 

To characterize structural waters and to follow the dynamics of reactions involving waters, it is necessary to be able to treat in detail the motions of these molecules. A methodology which includes solvents explicitly is required. Both conventional molecular dynamics techniques and the stochastic boundary molecular dynamics approaches can be used. When the region of 

Figuie 55. Stabilization of positively charged groups by bridging water molecules in the active site of native ribonuclease A without anions. The stereo drawing corresponds to a snapshot of the molecular dynamics trajectory at 15 ps. The picture includes only residues Lys-7, Arg-39, Lys-41, Lys-66, His-119, and the bound water molecules. The hydrogen-bonding criterion is the same as used in Fig. 54. 

In the active-site simulations of lysozyme108 (this chapter, Sect. B.2 above) similar water networks that stabilize charged groups have been observed. To illustrate the dynamics of the formation of such networks, a sequence of stereo plots showing the formation and evolution of a stable pair of positively charged residues is displayed in Fig. 56. The pair consists of (NH2)+ moieties of Arg-61 and Arg-73. The solvated structure evolved from a conformation obtained in a vacuum simulation of lysozyme.108,192 The sequence of plots shows the formation of the water-bridged pair over a time period from t = 0 ps to t 8 ps, which followed dynamical equilibration of the solvent around the fixed vacuum structure of the protein. After 8 ps, the ion-pair structure is stable, but fluctuations in the pattern of hydrogen bonds do occur typical 

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In an investigation of the role of water in enzymic catalysis. Brooks and Karplus (1989) chose lysozyme for their study. Stochastic boundary molecular dynamics methodology was applied, with which it was possible to focus on a small part of the overall system (i.e., the active site, substrate, and surrounding solvent). It was shown that both structure and dynamics are affected by solvent. These effects are mediated through solvation of polar residues, as well as stabilization of like-charged ion pairs. Conversely, the effects of the protein on solvent dynamics and... 

A metal ion cofactor acts as a Lewis acid in a variety of ways to help an enzyme catalyze a reaction. It can help bind the substrate to the active site of the enzyme it can complex with the substrate to increase its reactivity it can coordinate with groups on the enzyme, causing them to align in a way that facilitates the reaction and it can increase the nucleophilicity of water at the active site (Section 23.5). We have seen that Zn plays an important role in the hydrolysis reaction catalyzed by carboxypeptidase A (see page 1116). 

Subsequent to CO2 association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase II shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase II promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967 Lindskog and Coleman, 1973 Silverman and Lindskog, 1988). Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975). In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase II is a classical electrophilic catalyst that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C=0 in a precatalytic complex. 

CuZnSOD is a function dimer with each monomer containing one copper and one zinc in the active site bridged by the imidazole group of a histidine. The copper is also bound by three histidines and, in the oxidized state, a water molecule. In addition to the bridging histidines, the zinc is also bound by two histidines and an aspartate. Both oxidized and reduced enzymes are structurally similar except for a loss of the bond to the bridging histidines and a shift in position for the reduced copper. Copper is the only redox active metal zinc plays a structural role only. 

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