Probing of Active Site Residues of the Zinc Enzyme 5-Aminolevulinate Dehydratase by Spin and Fluorescence Labels

Abstract

5-Aminolevulinate dehydratase from bovine liver requires Zn(II) for its activity and is inhibited by micromolecular concentrations of Pb(II). To elucidate the structure of the active site and its interactions between the active site and the metal binding site we labeled the active site for fluorescence studies and ESR spectroscopy.o-Phthalaldehyde reacted with active site lysyl and cysteinyl residues to form a fluorescent isoindole derivative. The fluorescence energy was independent of the deprivation of Zn(II) and of its substitution by the inhibitory Pb(II). For ESR-studies five iodoacetamide and four isothiocyanate pyrrolidine-7V-oxyl derivatives with various spacer lengths were used to label the active site cysteinyl and lysyl residues, respectively. The ESR spectra of the modified enzyme preparations exhibited a significant immobilization of all labels, even with the longest spacers employed. Obviously the reactive cysteine is buried more than 12 A, and the active site lysine more than 11 A in a cleft of the enzyme structure. Zn(II) deprivation from the iodoacetamide spin-labeled enzyme caused a marked reversible increase in label mobility, whereas the Pb(II) substituted enzyme exhibited a smaller mobilization of the label. These results are interpreted by a model of the active site where the reactive cysteinyl and the lysyl side groups are close enough to be crosslinked by ophthalaldehyde within a distance of 3 A. A structural role is assigned to Zn(II) in the enzyme, since Zn(II) deprivation does not alter the fluorescence of the isoindole derivative and increases the mobility of the cysteine-bound spin labels in the active site cleft. Spin-und Fluoreszenzmarkierung. Untersuchung des aktiven Zentrums des Zink-Enzyms 5-Aminolävulinat-Dehydratase durch

Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase

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Haematological, hepatic and renal alterations after repeated oral or intraperitoneal administration of monoisoamyl DMSA. I. Changes in male rats

“…However, others support the assumption that zinc ions stabilize the active structure of the enzyme protein without directly interacting with the substrate. 35,36 The increase in blood zinc could be related to the increase in blood ZPP and the decrease in haemoglobin levels, suggesting mild anaemia and slightly altered haem synthesis. A marked depletion in the level of copper following MiADMSA administration by either route indicates that the compound has a greater complexing potential for copper.…”

Delta-aminolevulinate dehydratase activity in red blood cells of rats infected withTrypanosoma evansi

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