Biosynthesis of porphyrins and corrins. 2. Isolation, purification, and NMR investigations of the porphobilinogen deaminase covalent complex

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Abstract
The procedures for the generation of enzyme-substrate complexes from labeled porphobilinogens ([2,11-13C]PBG and [2,6,11-3H]PBG) with deaminase and the methods employed for their purification are described. Use of 13C NMR failed to detect the substrate bound to the enzyme, suggesting that the line width must be inordinately large. The complex was found to disproportionate with time when stored at 25.degree. C. However, enzyme-bound uroporphyrinogen I (uro''gen I) was detected, both in the intact protein and in the oligopeptides from tryptic digestion and peptide mapping. The first detection of an enzyme-substrate complex by 3H NMR is described for [3H]PBG and deaminase. The line widths of the observed resonances were found to be extremely large and dependent upon temperature, giving chemical shifts that suggest that involvement of a sulfhydryl group as the nucleophilic enzyme group that binds the substrate. The catalytic competence of this complex was also demonstrated by displacing bound [3H]PBG with unlabeled PBG. During the resultant formation of [3H]uro''gen I, a transient low-intensity signal was detected that has been tentatively assigned to the highly reactive azafulvene species, proposed in several mechanistic schemes for porphyrin biosynthesis.