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.

5-Aminolaevulinic acid dehydratase (ALAD) is an essential enzyme in most organisms, catalysing an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr-type condensation reaction of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole porphobilinogen. ALADs can be conveniently separated into two main groups: those requiring Zn2+ for activity (typified here by the enzymes from Escherichia coli and Saccharomyces cerevisiae, yeast) and those requiring Mg2+ (represented here by the enzyme from Pisum sativum, pea). Here we describe a detailed comparison of these two metal-dependent systems. Kinetically influential ionizations were identified by using pH-dependent kinetics. Groups with pKa values of approx. 7 and 10 (assigned to cysteine and lysine residues) were detected in the free enzyme and enzyme–substrate states of all three enzymes, and a further ionizable group with a pKa of approx. 8.5 (assigned to histidine) was found to be additionally important to the yeast enzyme. The importance of these residues was confirmed by using protein modifying reagents. Shifts in the pKa values of the pea and E. coli enzymes consequent on E–S complex formation suggest a change to a less hydrophobic microenvironment when substrate binds. Studies with inhibitors revealed that the three enzymes exhibit differential susceptibilities and, in the case of succinylacetone, this is reflected in Ki values that vary by three orders of magnitude. In addition, the crystallization of the yeast ALAD is described, raising the possibility of an X-ray-derived three-dimensional structure of this enzyme.

MAD experiments attempting to solve the structure of 5-aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insuf®cient for a complete structure solution but were invaluable in subsequent identi®cation of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn 2+ ion by Pb 2+ ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg cocrystal to provide the ®rst delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg cocrystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg 2+ had substituted for the same Zn 2+ cofactor ion as had Pb 2+ , a ®nding of fundamental importance for the understanding of mercury poisoning. In addition, Pt 2+ ions were found to bind at the same place in the structure. The re®ned structures of the Pband the Hg-complexed enzymes are presented at 2.5 and 3.0 A Ê resolution, respectively.

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RG42 6ET. 5-Aminolaevulinic acid dehydratase (ALAD; also called porphobilinogen synthase; EC 4.2.1.24) catalyses a key step in the tetrapyrrole biosynthetic pathway; the Knorr type condensation reaction between two molecules of 5-aminolaevulinic acid ( A M ) to form the monopyrrole, porphobilinogen. At present, the three-dimensional structure of this enzyme has not been determined although studies using electron microscopy and low angle X ray scattering have been reported [l, 21. This work describes the analysis of the structure of ALAD from yeast using circular dichmism (CD).Yeast ALAD was isolated and purified to homogeneity from E. coli JM109 cells overexpressing the yeast HEM2 gene as described in [3]. Protein characterisation revealed an octameric, zinc dependent, magnesium binding ALAD. Using JASCO spectrophotometers, CD spectra of the backbone and aromatic regions were obtained and analysis revealed yeast ALAD secondary structure to consist of approximately 33% a-helix and 21% p-pleated sheet.Yeast ALAD requires zinc for catalytic activity [3,4].However, when metal chelation was carried out using 1 ,lo-phenanthroline [5] and spectra were measured again, no changes in enzyme backbone or aromatic environment were observed, suggesting that metal removal from yeast ALAD does not grossly affect its structure. This is interesting as it has been suggested that one function of the metals in ALADs is to maintain structural integrity in the octameric enzymes [6J. The role of metals was investigated further using native PAGE of metal bound yeast and E. coli ALADs chelated with 1,lO phenanthroline or EDTA. Results revealed predominantly a single band for both metalbound enzymes but on metal chelation, a laddering of bands was observed for E. coli L A D but not that from yeast. It therefore appears that metals in yeast ALAD are unlikely to fill a major structural role although such a function is suggested for E. coli ALAD [6]. CD studies with the denaturants urea and sodium dodecyl sulphate suggest that the yeast ALAD structure is remarkably stable to disruption.Similarly,CD was used to study yeast ALAD structure in the presence of increasing stoichiometric ratios of ALA. No changes in either spectral region were noted. suggesting that conformational changes required during catalysis are small. This is in accordance with conclusions drawn from the study of the enzyme's pH dependent kinetics [3].Thermal denaturation of yeast ALAD was followed in the backbone region. The melting curve showed a temperature sensitive enzyme with two stage irreversible denaturation. The first stage was quick q,I, = 49.2OC) whereas the second stage was slower (Tm = 60.2oC). For comparison, the experiment was repeated with E. coli ALAD, and this also showed two stage melting with a slower first phase qm=61.60C) and fast second stage vm=64.50C). Differences in the arrangement of structural motifs are therefore anticipated between the two enzymes.Towards the goal of a three-dimensional structure for the enzyme, crystallisation of both native an...