5-Aminolaevulinate dehydratase (ALAD) is a homo-octameric metallo-enzyme that catalyses the formation of porphobilinogen from 5-aminolaevulinic acid. The structure of the yeast enzyme has been solved to 2.3 A resolution, revealing that each subunit adopts a TIM barrel fold with a 39 residue N-terminal arm. Pairs of monomers wrap their arms around each other to form compact dimers and these associate to form a 422 symmetric octamer. All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263), one of which, Lys 263, forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites one of which is formed by three cysteine residues (133, 135 and 143) while the other involves Cys 234 and His 142. ALAD has features at its active site that are common to both metallo- and Schiff base-aldolases and therefore represents an intriguing combination of both classes of enzyme. Lead ions, which inhibit ALAD potently, replace the zinc bound to the enzyme's unique triple-cysteine site.

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Aspartic proteinases are a class of enzymes widely distributed among fungi, plants, vertebrates, and viruses. They are involved in numerous disease conditions, including hypertension, amyloid disease, malaria, and AIDS. 1 In HIV, the proteinase is essential for the maturation of the virus particle, and inhibitors have a proven therapeutic effect in the treatment of AIDS. There have also been significant recent advances in the development of orally active renin inhibitors which are in clinical trials. 2 Thus inhibitors to this class of enzyme with improved characteristics are therefore much sought after as potential therapeutic agents. The aspartic proteinase class of enzyme comprises two structurally similar domains, each contributing an aspartic acid residue to form a catalytic dyad that cleaves the substrate peptide bond. The two catalytic aspartate carboxyls are held coplanar by a network of hydrogen bonds involving the surrounding main chain and the conserved amino acid side-chain groups. Most aspartic proteinases are inhibited by pepstatin, a microbial peptide that contains the unusual amino acid statine, and a number of crystal structures of complexes containing this naturally occurring analogue have been reported. 3,4 Endothiapepsin, a member of the aspartic proteinase family, is composed of 330 amino acid residues, with roughly 170 residues in each domain. Endothiapepsin is derived from the fungus Endothia parasitica, and numerous structures of this enzyme bound to a range of renin inhibitors have been analyzed with the aim of developing improved compounds by structure-based design. 5 Crystallographic studies of all native aspartic proteinases have revealed a water molecule hydrogen bonded to the carboxyl groups of both aspartate residues. This water molecule is within hydrogen-bonding distance of all four carboxyl O atoms and has been implicated in catalysis. It has been suggested 6 that this water is partly displaced upon substrate binding and is polarized by one of the catalytic aspartate residues. It then initiates enzymatic catalysis by attacking the scissile bond carbonyl group of the substrate. The transition state of aspartic proteinase catalysis has been studied most extensively using enzyme-inhibitor complexes which possess a range of different transition state mimics. Most inhibitors mimic one or both hydroxyls in the putative transition state. The statine-based inhibitors contain one hydroxyl that occupies the same position as the water E-mail: coatesl@ornl.gov. † Oak Ridge National Laboratory. ‡ University of Toledo. § Los Alamos National Laboratory. ¶ University College London.Supporting Information Available: Data collection and refinement statistics for all three structures discussed within this paper, the atomic resolution, the room temperature X-ray, and room temperature neutron. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 1. NIH Public AccessResults from this study suggest that the transition state is stabilized by a negative charge on Asp...