Enzymatic nucleophiles

In their studies the enzyme activity decreased when reagents were added that complex with thiols hence it was concluded that cysteine was probably the key enzymatic nucleophile. The production of this enzyme could be induced by exposure of the bacteria to various phenylamide- or phenylurea-containing herbicides and fungicides. The enzyme was also capable of hydrolyzing a variety of other phenylamides and phenylureas, albeit at somewhat different rates. 

From the structure of this compound, you see a good leaving group in the chloride, Cl, on several parts of this compound s structure. But since the chlorines are attached to a carbon that is also substituted with three other large groups, or they are on aryl carbons, they cannot be approached readily from the back side by an enzymatic nucleophile as used in microbial hydrolases. Hence, we do not expect microorganisms to use a nucleophilic attack and a hydrolysis approach. 

A considerable amount of work, both with thymidylate synthetase itself and model systems, indicates that in the mechanism of thymidylate synthetase a key step is the attack of an enzymatic nucleophile (believed to be cysteine) on C-6 of dUMP to give a 5,6-dihydro-dUMP intermediate (B-77MI11003). A number of observations are in accord with such a nucleophilic catalysis by the enzyme. Among these are the demonstration that thymidylate synthetase, in the absence of the cofactor, catalyzes the exchange of tritium from [5-3H]dUMP for protons of water (79B2794), and the recent isolation of a covalent adduct formed between 5-nitro-dUMP and thymidylate synthetase (80JBC(255)5538 see also 80MI11003). 

Scheme 2 Two potential reaction mechanisms for phosphatases or sulfatases are shown here using a phosphate ester. In (a), the phosphoryl group is transferred directly to a water molecule, which is typically bound to one or two metal ions if the substrate is made chiral at phosphorus, the stereochemical outcome is inversion. In (b), the phosphoryl group is first transferred to an enzymatic nucleophile E-POs " is a covalent phosphoenzyme intermediate. In a subsequent step, this intermediate is hydrolyzed. Since each step occurs with inversion of configuration at phosphorus, the net outcome is retention. The same principles apply to sulfuryl transfer. P, = inorganic phosphate.

The enzymatic nucleophile is similar in kind and reactivity to the ultimate solution acceptor. Examples of this class include the serine proteases and the alkahne phosphatase. The serine hydroxyl group is similar in chemical reactivity to the hydroxyl group of water, the final acceptor in these group transfer reactions (Fersht, 1985). For example, the active site Ser200 and His444 of cholinesterase are involved in a putative catalytic triad to effect acyl transfer (Taylor, 1991) ... 

The enzymatic nucleophile is intrinsically more reactive than the ultimate acceptor. Several enzymes use the imdazole side chain of His as a transient acceptor in phosphoryl transfer reaction (Fersht, 1985). Amines react faster than alcohol with phosphoryl compounds, yet they form less stable adducts that can therefore readily allow rapid turnover. 

Formation of a covalent adduct with an enzymatic nucleophile increases the reactivity of the substrate, which facilitates a reaction that is distinct from adduct formation. Examples of this include the thymidylate synthetase, in which the sulfhydryl of an active site Cys adds to C6 of the uracil. This breaks the aromaticity and adds electron density to increase the nucleophilicity of C5, thereby facilitating transfer of a methylene equivalent from tetrahydrofolate (Carreras and Santi, 1995). 

Ghlorobenzoic acid is a degradation product of some RGBs. It is now known that certain bacteria are able to dehalogenate 4-chlorobenzoic acid by an enzymatic nucleophilic aromatic substitution reaction. The product is 4-hydroxybenzoic acid, and a mechanism for this enzyme-catalyzed process is shown here. The sequence begins with the thioester of 4-chlorobenzoic acid derived from coenzyme A (GoA) ... 

Enzymatic reactions are the most vividly studied phenomena in organic chemistry and biochemistry. Such studies concern the mechanistic and enzyme-structural aspects. Since the 1990s many results of 3D structure analysis of enzymes have become available. For example, Fig. 4 illustrates the structure of the catalytic core domain of the family 5 endoglucanase, which suggests that the catalytic acid/base and enzymatic nucleophile are due to carboxylic acid residues Glu 139 and Glu 228, respectively [32]. In the catalytic site, cellotriose is included for reference. 

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