Nucleophilic groups enzymes

Altman and coworkers (Scheme 4). Cyclopropane formation assisted by the participation of enzyme nucleophilic groups was proposed by van Tamelen and Schwartz (Scheme 5), Beytia and coworkers (Scheme 6), and the Utah groups(Scheme 7). 

Clavulanic acid is a mechanism-based irreversible inhibitor and could be classed as a suicide substrate (Chapter 4). The drug fits the active site of (3-lactamase and the 13-lactam ring is opened by a serine residue in the same manner as penicillin. However, the acyl-enzyme intermediate then reacts further with another enzymic nucleophilic group (possibly NH2) to bind the drug irreversibly to the enzyme (Fig. 10.54). The mechanism requires the loss or gain of protons at various stages and an amino acid such as histidine present in the active site would be capable of acting as a proton donor/acceptor (compare the mechanism of acetylcholinesterase in Chapter 11). 

An enzyme nucleophilic group, most likely SH in the context of the Pro-Cys doublet conserved in all known m C-MTases, is believed to assist the chemical catalysis by attacking the C-6 of the substrate cytosine. Formation of a covalent intermediate apparently renders the otherwise inert C-5 electron rich and facilitates the electrophilic transfer of a methyl group from AdoMet. In the absence of AdoMet, M Hhal catalyzes the exchange of the 5-H of the substrate cytosine for protons of water at a rate about sevenfold higher than the rate of methylation. 

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

Q An amino acid reacts with the enzyme-bound PLP inline by nucleophilic addition of its -NH2 group to the C=N bond of the imine, giving a PLP-amino acid imine and releasing the enzyme amino group. 

A major question has been that of bifunctional catalysis . For example, if a micelle contains both nucleophilic groups and groups which can transfer protons one might hope to achieve high rates of deacylation by having concerted nucleophilic attack and proton transfer (Scheme 6). Such concerted processes are well established in enzymic reactions, but evidence in... 

Rauter s group exploited the synthesis of sugar derived bicyclic butenolides ( e.g. 140, Fig. 44),60 which possess cytotoxic and antitumor activities. The key structural feature of such compounds consists of the presence of the a,(3-unsaturated lactone, which allows them to act as Michael acceptors for the addition of enzymes nucleophiles. 

Allenic amino acids belong to the classical suicide substrates for the irreversible mechanism-based inhibition of enzymes [5], Among the different types of allenic substrates used for enzyme inhibition [128, 129], the deactivation of vitamin B6 (pyr-idoxal phosphate)-dependent decarboxylases by a-allenic a-amino acids plays an important role (Scheme 18.45). In analogy with the corresponding activity of other /3,y-unsaturated amino acids [102,130], it is assumed that the allenic amino acid 139 reacts with the decarboxylase 138 to furnish the imine 140, which is transformed into a Michael acceptor of type 141 by decarboxylation or deprotonation. Subsequent attack of a suitable nucleophilic group of the active site then leads to inhibition of the decarboxylase by irreversible formation of the adduct 142 [131,132]. 

However, mechanistic experiments have involved the apices of the cyclopropane ring as targets for the nucleophilic group of the enzyme. Only the pro-R hydrogen was usually removed by the enzyme and transferred to NAD+, and the stereochemical course of the nucleophilic ring opening of the cyclopropanes was consistent with predictions on the basis of frontier orbital theory prediction [48]. 

Host structures that, like many hydrolytic enzymes [16], contain an OH/SH group that acts as acyl-receiving/acyl-releasing unit have been reported by several authors [18]. Such host structures are acylated by suitably functionalized esters at enhanced rates thanks to the presence of tailored binding sites proximal to the nucleophilic group. However, since no provision was made to activate the cleavage ofthe acylated host, many of the investigated systems showed little or no turnover capability. 

In the presence of a covalent catalyst (an enzyme with a nucleophilic group X ) the reaction becomes... 

Double-displacement mechanisms. In a doubledisplacement mechanism sucrose phosphorylase would catalyze two consecutive single displacements, each with inversion. A nucleophilic group of the enzyme would react in Eq. 12-7, step a. In step b, a phosphate would react to regenerate the enzyme with its free nucleophilic group -B. ... 

For acid-catalyzed hydrolysis of methyl glucosides53 the kinetic isotope effect observed for the oxygen of the leaving group was /c,6()//c18() = 1.024-1.026. Observation of similar effects for enzymes supports the participation of an acidic group of the protein (Glu 35 of lysozyme) in catalysis but does not eliminate the possibility of concerted involvement of a nucleophilic group, e.g., Asp 52 in lysozyme.81 82... 

Enzymatic reactions involving addition to a C = C bond adjacent to a carbonyl group (or in which elimination occurs a,P to a carbonyl) are numerous. Except for some enzymes acting by free radical mechanisms, the nucleophilic group always adds at the P position suggesting that the mechanism portrayed by Eq. 13-7 is probable. It is noteworthy that frequently in a metabolic sequence a carbonyl group is deliberately introduced to... 

Notice the stereochemistry of Eq. 13-18, step a. Orbital interaction rules predict that if the elimination is a concerted process it should be syn. The observed anti elimination suggests a more complex mechanism involving participation of a nucleophilic group of the enzyme.97... 

Nucleophilic groups from enzymes can add to double bonds, e.g., in an aminoacrylate Schiff base, or to multiple bonds present in die inhibitor. An example is y-vinyl y-aminobutyrate (4-amino-5-hexenoic acid), another inhibitor of brain y-aminobutyrate aminotransferase which is a useful anticonvulsant drug. 

These compounds act as alkylating agents N2 is released and a nucleophilic group from the enzyme becomes attached at the carbon atom indicated.134 Other inhibitors bind noncovalently to form dead-end complexes.1343... 

Some data suggested that a transient covalent linkage of tRNA to the synthetases may form through addition of a nucleophilic group of the enzyme to the 6 position of the uracil (or 4-thiouracil) present in position 8 of all tRNAs (Eq. 19-3).257 The two isoacceptors tRNATyr species in E. coli contain 4-thiouracil at this position. The C=C bond in this base can be saturated by sodium borohydride reduction, which was found not only to prevent the covalent interaction with the enzyme but also to prevent aminoacylation of the tRNA. However, Eq. 29-3 probably describes a side reaction irrelevant to tRNA function. 

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