Characterization of a Recombinant Pea 5-Aminolevulinic Acid Dehydratase and Comparative Inhibition Studies with the Escherichia coli Dehydratase

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• 作者：Kwai-Ming Cheung ; Paul Spencer ; Michael P. Timko ; and Peter M. Shoolingin-Jordan
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：February 4, 1997
• 年：1997
• 卷：36
• 期：5
• 页码：1148 - 1156
• 全文大小：216K
• 年卷期：v.36,no.5(February 4, 1997)
• ISSN：1520-4995

文摘

Pea 5-aminolevulinic acid dehydratase (ALAD) was purified 200-foldfrom a recombinantoverproducing strain of Escherichia coli, yielding anoctameric enzyme with a specific activity of 280units mg^-1. Divalent metal ions wereessential, Mg²⁺, Mn²⁺, andCo²⁺ ions all supporting activity, whereasZn²⁺ ions could not. Equilibrium dialysis and atomicabsorption studies revealed two Mg²⁺ ionbindingsites per subunit. Pea ALAD bound the substrate 5-aminolevulinicacid covalently through a Schiff baseat the P-site, electrospray mass spectrometry of the reducedenzyme-ALA Schiff base complex showingthe presence of one P-site per subunit. The amino acid residuemodified by ALA was identified byMALDI-MS and Edman sequencing as Lys-293, analogous to the active siteLys-247 of E. coli ALADand Lys-252 of mammalian ALAD. Comparative studies of pea ALADwith E. coli ALAD using theinhibitors 3-acetyl-4-oxoheptane-1,7-dioic acid (AOHD) andsuccinylacetone (SA) indicated similar modesof inhibition, with the formation of a Schiff base complex between theinhibitors and the active sitelysine. Studies with the ALA homolog, 4-amino-3-oxobutanoic acid(AOB), revealed that it is specificfor the A-site of both the pea and E. coli ALADs. Aninteresting difference exists between the enzymes,however, pea ALAD being far more susceptible to inhibition with AOBthan the E. coli enzyme. AOBbound 10 times better to the A-site of pea ALAD compared to thesubstrate, ALA. Despite the 2000times lower K_i of AOB for pea ALAD, no abortiveSchiff base intermediate, between enzyme-boundALA at the P-site and AOB bound at the A-site, could bedemonstrated.