Enzyme active sites, water role

Blevins and Tulinsky (1985) suggested two functions for the solvent at the chymotrypsin active site (1) solvation of the Asp—His—Ser catalytic triad, and (2) a guiding effect on the substrate in formation of the enzyme-substrate complex, provided by several waters at the end of the specificity site. X-Ray diffraction results have suggested a role of active-site water in determining the kinetics or equilibria of substrate binding for other proteins (Section IV). 

Penicillin G acylase (PGA) has pivotal role in industry for the synthesis of penicillin antibiotics. PGA catalyzes the hydrolysis of peniciUm and other P-lactam antibiotics to produce 6-amino penicillinic acid [53, 54]. Stability of PGA was investigated by assaying the enzyme activity at different time points after incubation of PGA in various ILs [53]. In the absence of substrate, about 2,000-fold increase in t, was observed in a hydrophobic IL, [EMIM][Tf2N] with respect to twpropanol. Whereas in the presence of substrate, PGA showed less stability in [Tf N]" containing ILs. The reverse trend was found for PGA in a water miscible IL [BMIM][PFg], i.e., PGA showed 9 times increase in tj in [BMIM][PF ] in the presence of substrate even at an elevated temperature of 40°C [54]. These altered t, values in hydrophobic ILs in the presence of substrates were explained on the basis of cumulative outcome of specific interaction of substrates with the active site of enzyme and inhibitory effect of the hydrolytic products formed in the reaction medium. It can be speculated that in a more hydrophilic IL like [BMIM][PF ], substrates shield the enzyme active site from direct interaction with the ionic matrix and thus imparts a stabilizing effect whereas in hydrophobic IL [EMIM][TfjN], the inhibitory effect of hydrolytic products predominates and destabilizes the enzyme. 

In many enzymes, a substantial part of the protein itself plays a significant role in the chemical reaction. Along with the substrate, any required cofactors, and sometimes active-site water molecules, certain amino acid residues must be described quantum mechanically. Inclusion of residues in the QM subsystem requires one or more covalent bonds across the QM/MM boundary. The bond between the Ca and Cp atoms of an amino acid is a popular choice for the QM/MM interface, as it is a reasonably well- behaved, nonpolar bond, and it is often sufficiently far away from the reactive center. 

Y.-M. Zhang, J. Hurlbert, S.W. White, C.O. Rock, Roles of the active site water, histidine 303, and phenylalanine 396 in the catalytic mechanism of the elongation condensing enzyme of Streptococcus pneumoniae. J. Biol. Chem. 281, 17390-17399 (2006)... 

We begin this overview of manganese biochemistry with a brief account of its role in the detoxification of free radicals, before considering the function of a dinuclear Mn(II) active site in the important eukaryotic urea cycle enzyme arginase. We then pass in review a few microbial Mn-containing enzymes involved in intermediary metabolism, and conclude with the very exciting recent results on the structure and function of the catalytic manganese cluster involved in the photosynthetic oxidation of water. 

At this time, the proposal of additional access channels is quite conjectural. It seems likely that there is a channel or access route to the proximal side of the heme in order to provide access for the hydrogen peroxide or water needed for heme oxidation and His-Tyr bond formation. Furthermore, the electron density of compoimd I from PMC (97) reveals the presence of an anionic species that is not present in the native enz5une. However, the rapid influx-efflux rates up to 10 per sec needed for such a species to be a component of compoimd I would pose interesting constraints on a channel, and there does not seem to be a likely candidate in the region. Similarly, the potential channel leading to the cavity at the molecular center is not an ideal candidate for substrate or product movement because of its relationship to the active site residues. However, if the lateral channel is truly blocked by NADPH in small-subunit enzymes, this route may provide an alternative access or exhaust route. Both of these latter two channels require further investigation before a clear role can be ascribed to them. 

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. 

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