The arrangement of ligands around the zinc ions includes a water molecule, presumably in the form of a hydroxide ion, coordinated to both metal ions. This hydroxide ion is situated 2.9 A from the carbonyl carbon of the substrate in such a position that it could act as the nucleophile during catalysis. The reaction mechanism may also have implications for the action of metallo-beta-lactamases.

A sequence encoding a novel glutathione transferase, GST A4-4, has been identified in a human fetal brain cDNA library. The protein has been produced in Escherichia coli after optimization of the codon usage for high-level heterologous expression. The dimeric protein has a subunit molecular mass of 25704 Da based on the deduced amino acid composition. Human GST A4-4 is a member of the Alpha class but shows only 53% amino acid sequence identity with the major liver enzyme GST A1-1. High catalytic efficiency with 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation is a significant feature of GST A4-4. The kcat/Km values for 4-hydroxynonenal and 4-hydroxydecenal are > 3 x 10(6) M-1. s-1, several orders of magnitude higher than the values for conventional GST substrates. 4-Hydroxynonenal and other reactive electrophiles produced by oxidative metabolism have been linked to aging, atherosclerosis, cataract formation, Parkinson's disease and Alzheimer's disease, as well as other degenerative human conditions, suggesting that human GST A4-4 fulfills an important protective role and that variations in its expression may have significant pathophysiological consequences.

Glyoxalase I (EC 4.4.1.5) catalyses the isomerization Sweden of the thiohemiacetal of glutathione (GSH) and a 2 Corresponding author 2-oxoaldehyde into the thiolester of GSH and the corresponding 2-hydroxycarboxylic acid. The substrate of the The zinc metalloenzyme glyoxalase I catalyses the reaction, the thiohemiacetal, is formed non-enzymatically. glutathione-dependent inactivation of toxic methylGlyoxalase II then hydrolyses the thiolester to produce glyoxal. The structure of the dimeric human enzyme GSH and a free 2-hydroxycarboxylic acid. Human in complex with S-benzyl-glutathione has been deterglyoxalase I is a dimer of 43 kDa containing 183 amino mined by multiple isomorphous replacement (MIR) acid residues per monomer.

Amodiaquine (AQ) metabolism to N-desethylamodiaquine (DEAQ) is the principal route of disposition in humans. Using human liver microsomes and two sets of recombinant human cytochrome P450 isoforms (from lymphoblastoids and yeast) we performed studies to identify the CYP isoform(s) involved in the metabolism of AQ. CYP2C8 was the main hepatic isoform that cleared AQ and catalyzed the formation of DEAQ. The extrahepatic P450s, 1A1 and 1B1, also cleared AQ and catalyzed the formation of an unknown metabolite M2. The K m and V max values for AQ N-desethylation were 1.2 M and 2.6 pmol/ min/pmol of CYP2C8 for recombinant CYP2C8, and 2.4 M and 1462 pmol/min/mg of protein for human liver microsomes (HLMs), respectively. Relative contribution of CYP2C8 in the formation of DEAQ was estimated at 100% using the relative activity factor method. Correlation analyses between AQ metabolism and the activities of eight hepatic P450s were made on 10 different HLM samples. Both the formation of DEAQ and the clearance of AQ showed excellent correlations (r 2 ϭ 0.98 and 0.95) with 6␣-hydroxylation of paclitaxel, a marker substrate for CYP2C8. The inhibition of DEAQ formation by quercetin was competitive with K i values of 1.96 for CYP2C8 and 1.56 M for HLMs. Docking of AQ into the active site homology models of the CYP2C isoforms showed favorable interactions with CYP2C8, which supported the likelihood of an N-desethylation reaction. These data show that CYP2C8 is the main hepatic isoform responsible for the metabolism of AQ. The specificity, high affinity, and high turnover make AQ desethylation an excellent marker reaction for CYP2C8 activity.Amodiaquine (AQ) is a 4-aminoquinoline derivative that has been widely used for treatment of malaria over the past 50 years. It is intrinsically more active than the other 4-aminoquinoline, chloroquine, against Plasmodium falciparum parasites, which are moderately chloroquine resistant. The drug is therefore increasingly being considered as a replacement for chloroquine as a first line drug in Africa because of widespread chloroquine resistance. Because of major side effects, mainly agranulocytosis, observed during prophylactic use of the drug, AQ is now only recommended for treatment of malaria, for which no serious cases of toxicity have been reported (Laurent et al., 1993).Upon oral administration, AQ is rapidly absorbed and extensively metabolized such that very little of the parent drug is detected in the plasma. The main metabolite of AQ is N-desethylamodiaquine (DEAQ) with other minor metabolites being 2-hydroxyl-DEAQ and N-bisdesethylAQ (bis-DEAQ) (Churchill et al., 1985Mount et al., 1986). Whereas the formation of DEAQ is rapid, its elimination is very slow with a terminal half-life of over 100 h (Winstanley et al., 1987;Laurent et al., 1993). AQ and DEAQ both have antimalarial activity, but AQ is 3 times more active (Churchill et al., 1985). However, since AQ is rapidly cleared and the formed DEAQ attains high plasma concentrations for a long time, AQ is considered a prodrug,...

The structures of human glyoxalase I in complexes with S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione (HIPC-GSH) and S-p-nitrobenzyloxycarbonylglutathione (NBC-GSH) have been determined at 2.0 and 1.72 A resolution, respectively. HIPC-GSH is a transition state analogue mimicking the enediolate intermediate that forms along the reaction pathway of glyoxalase I. In the structure, the hydroxycarbamoyl function is directly coordinated to the active site zinc ion. In contrast, the equivalent group in the NBC-GSH complex is approximately 6 A from the metal in a conformation that may resemble the product complex with S-D-lactoylglutathione. In this complex, two water molecules occupy the liganding positions at the zinc ion occupied by the hydroxycarbamoyl function in the enediolate analogue complex. Coordination of the transition state analogue to the metal enables a loop to close down over the active site, relative to its position in the product-like structure, allowing the glycine residue of the glutathione moiety to hydrogen bond with the protein. The structure of the complex with the enediolate analogue supports an "inner sphere mechanism" in which the GSH-methylglyoxal thiohemiacetal substrate is converted to product via a cis-enediolate intermediate. The zinc ion is envisioned to play an electrophilic role in catalysis by directly coordinating this intermediate. In addition, the carboxyl of Glu 172 is proposed to be displaced from the inner coordination sphere of the metal ion during substrate binding, thus allowing this group to facilitate proton transfer between the adjacent carbon atoms of the substrate. This proposal is supported by the observation that in the complex with the enediolate analogue the carboxyl group of Glu 172 is 3.3 A from the metal and is in an ideal position for reprotonation of the transition state intermediate. In contrast, Glu 172 is directly coordinated to the zinc ion in the complexes with S-benzylglutathione and with NBC-GSH.