Deprotonation

-B-ENZYME

Nucleophilic attack Fig. 7.21 Schematic showing mechanism of glucuronidation reactions.

7.6 Enzymes Catalysing Drug Conjugation | 91

Similar mechanisms apply to sulphate transferases in which the donor is 3'-phos-phoadenosine-5-phosphosulphate (PAPS). The accepting groups in the molecule are phenols, alcohols and hydroxylamines. The sulphotransferases are relatively nonspecific, however, phenol-sulphotransferase is probably the most relevant to the medicinal chemist. The similarity in mechanism [17, 18] is shown by comparing the Vmax values for glucuronyl transferase and sulphotransferase for a series of power substituted phenols. Figure 7.22 shows the log Vmax for these series plotted against the Hammett sigma value.

Fig. 7.22 Relationship between sigma value and enzyme rate for glucuronyl and sulphotransferases indicating the role of nucleophilicity.

Fig. 7.22 Relationship between sigma value and enzyme rate for glucuronyl and sulphotransferases indicating the role of nucleophilicity.

The negative slope of both curves indicates the greater the nucleophilicity (electron-donating ability) of the phenolate anion the faster the rate of the reaction. The initial deprotonation of the phenol is apparently not rate limiting but must occur rapidly so that those compounds with high pKa values can be deprotonated. Recently X-ray crystallography data [19, 20] has been obtained for various sulphotransferases. The active site comprises a hydrophobic pocket formed by phenylalanine residues (see Figure 7.23). In the case of the catecholtransferase SULT1A3, a glutamic acid residue provides an ion pair interaction with the basic nitrogen of many of its natural substrates such as dopamine. A critical residue in the catalytic process is a lysine which stabilizes the transition state and via a hydrogen bond interaction with the bridge oxygen, between the 5'-phosphate group and the sulphate group of PAPS, acts as a catalytic acid to enhance the dissociative nature of the sulphuryl transfer mechanism. The other critical residue is an histidine, which acts as the base which depro-tonates the phenol (or other group) to a phenoxide. The resultant nucleophile can then attack the sulphur atom of the transferring sulphuryl group.

The glucuronide and sulphotransferases are present in the gut as well as the liver and catalyze the metabolism of many phenol- or catechol-containing drugs (morphine, isoprenaline etc.) during their passage through the gut. The ready conjugation of phenolic functions by both glucuronyl and sulphotransferase systems means that drugs such as morphine are cleared as both glucuronide and sulphate metabolites.

Fig. 7.23 Schematic of the active site of strate. Leu 48 stabilizes the transition state catechol sulphotransferase (SULT1A3). with and His 108 deprotonates one of the catechol dopamine in the active site. Phe 142, 81 and hydroxyls to form the phenoxide nucleophile

24 form a hydrophobic pocket whilst Glu 146 to allow the reaction to proceed. provides an ion pair interaction with the sub-

Fig. 7.23 Schematic of the active site of strate. Leu 48 stabilizes the transition state catechol sulphotransferase (SULT1A3). with and His 108 deprotonates one of the catechol dopamine in the active site. Phe 142, 81 and hydroxyls to form the phenoxide nucleophile

24 form a hydrophobic pocket whilst Glu 146 to allow the reaction to proceed. provides an ion pair interaction with the sub-

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