Porphobilinogen Synthase from Pea: Expression from an Artificial Gene, Kinetic Characterization, and Novel Implications for Subunit Interactions

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• 作者：Jukka Kervinen ; Roland L. Dunbrack ; Jr. ; Samuel Litwin ; Jacob Martins ; Robert C. Scarrow ; Marina Volin ; Anthony T. Yeung ; Erica Yoon ; and Eileen K. Jaffe
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 1, 2000
• 年：2000
• 卷：39
• 期：30
• 页码：9018 - 9029
• 全文大小：271K
• 年卷期：v.39,no.30(August 1, 2000)
• ISSN：1520-4995

文摘

Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles suchas heme, chlorophyll, and vitamin B₁₂. The homooctameric metalloenzyme catalyzes the condensation oftwo 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen. An artificialgene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems duringbacterial expression caused by suboptimal codon usage and was constructed by recursive polymerasechain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putativechloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specificactivity is protein concentration dependent, which indicates that a maximally active octamer can dissociateinto less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pK_a valuesof 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit;kinetic data support two functionally distinct types of Mg(II) and the third appears to be nonphysiologicand inhibitory. Analysis of the protein concentration dependence of the specific activity suggests that theminimal functional unit is a tetramer. A model of octameric pea PBGS was built to predict the locationof intermolecular disulfide linkages that were revealed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As verified by site-specific mutagenesis, disulfide linkages can formbetween four cysteines per octamer, each located five amino acids from the C-terminus. These data areconsistent with the protein undergoing conformational changes and the idea that whole-body motion canoccur between subunits.