The Enzymatic Synthesis Of Porphobilinogen

ALAD catalyzes the first of three steps for the transformation of ALA into uropor-phyrinogen III, which are found in all living organisms that synthesize tetrapyrroles (13). The enzymes exist as homo-octamers with subunit molecular masses of 35 to 45 kDa, depending on the source organism, and catalyze the condensation of two molecules of ALA into the pyrrole PBG (Figure 3). Comparisons between the amino acid sequences derived from nucleotide sequencing indicate that the enzyme structure is strongly conserved, and this is confirmed by crystallographic studies that show that both prokaryotic and eukaryotic dehy-dratases have a similar (¬ęP)g barrel subunit structure (7,8). The active site is located at the center of the barrel with two juxtaposed lysines and an aspartic acid playing essential roles in catalysis. One of the lysines, K247 in the E. coli enzyme, forms a Schiff base with the substrate molecule at the P-site, so called because it binds the ALA molecule that ultimately becomes the propionic acid side chain of the product PBG. Pairs of

Figure 3. The biosynthesis of porphobilinogen from 2 molecules of ALA. It has been established that the proR hydrogen of the ALA molecule occupying the P (propionate) site is stereoselectively removed during the reaction.

subunits are arranged as dimers, held together by long N-terminal arms, with four dimers arranged in D4 symmetry to form the octamer. The conservation of the quaternary structure through evolution may, in part, be as a consequence of a second and somewhat surprising function of the protein, namely, as the inhibitory complex of the proteasome (10).

ALADs differ in their requirement for divalent metal ions. Those found in animals require only zinc for activity, those found in plants require only magnesium, and others require zinc but are activated by magnesium (14). E. coli ALAD, used for the methods below, is of the magnesium-activated zinc type. The metal ion in the zinc-dependent enzymes is chelated to a triple cysteine motif at the active site and appears to be an essential part of the active site that binds the second molecule of ALA at the A-site. The zinc-dependent dehy-dratases are exceptionally sensitive to low levels of lead, which displaces the zinc ion and inactivates the enzyme. Although ALADs may be purified from a variety of natural sources, the most convenient purification (31) is from a recombinant strain of E. coli harboring the E. coli hemB gene.

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