Porphobilinogen synthase from pea: Expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions

Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin Bit. The homooctameri c metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid mol ecules to form the tetrapyrrole precursor porphobilinogen, An artificial ge ne encoding PEGS of pea (Pisum sativum L.) was designed to overcome previou s problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic olig onucleotides. The recombinant 330 residue enzyme without a putative chlorop last transit peptide was expressed in Escherichia coli and purified in 100- mg quantities. The specific activity is protein concentration dependent, wh ich indicates that a maximally active octamer can dissociate into less acti ve smaller units. The enzyme is most active at slightly alkaline pH; it sho ws two pK(a) values of 7.4 and 9.7. Atomic absorption spectroscopy shows ma ximal binding of three Mg(II) per subunit; kinetic data support two functio nally distinct types of Mg(II) and the third appears to be nonphysiologic a nd inhibitory. Analysis of the protein concentration dependence of the spec ific activity suggests that the minimal functional unit is a tetramer. A mo del of octameric pea PEGS was built to predict the location of intermolecul ar disulfide linkages that were revealed by nonreducing sodium dodecyl sulf ate-polyacrylamide gel electrophoresis. As verified by site-specific mutage nesis, disulfide linkages can form between four cysteines per octamer, each located five amino acids from the C-terminus. These data are consistent wi th the protein undergoing conformational changes and the idea that whole-bo dy motion can occur between subunits.