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Publication Detail
Comparative studies on the 5-aminolaevulinic acid dehydratases from Pisum sativum, Escherichia coli and Saccharomyces cerevisiae.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Comparative Study
  • Authors:
    Senior NM, Brocklehurst K, Cooper JB, Wood SP, Erskine P, Shoolingin-Jordan PM, Thomas PG, Warren MJ
  • Publication date:
    01/12/1996
  • Pagination:
    401, 412
  • Journal:
    Biochem J
  • Volume:
    320 ( Pt 2)
  • Status:
    Published
  • Country:
    England
  • Print ISSN:
    0264-6021
  • Language:
    eng
  • Keywords:
    Amino Acid Sequence, Animals, Cattle, Cloning, Molecular, Crystallization, Enzyme Inhibitors, Escherichia coli, Humans, Hydrogen-Ion Concentration, Kinetics, Magnesium, Mice, Molecular Sequence Data, Mutagenesis, Insertional, Peas, Plasmids, Polymerase Chain Reaction, Porphobilinogen Synthase, Rats, Recombinant Proteins, Saccharomyces cerevisiae, Schiff Bases, Sequence Homology, Amino Acid, Spectrophotometry, Atomic, Zinc
Abstract
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 micro-environment 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.
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