Mechanism of porphobilinogen synthase. Possible role of essential thiol groups.

The inactivation of beef liver porphobilinogen synthase by the alkylating agents iodoacetate and iodoacetamide has been investigated. The synthase can be inactivated by these reagents only if the enzyme has been activated initially by sulfhydryl reducing agents. Inactivation by both iodoacetate and iodoacetamide was the result of modification of cysteinyl residues. A plot of the incorporation of W-labeled alkylating reagents uersus activity extrapolated to the modification of approximately one fast reacting cysteinyl residue per monomer (M, = 35,000) as responsible for the loss of activity by either iodoacetate or iodoacetamide; the pH of half-maximum alkylation, with both reagents, was about 5.2 indicating that each reagent alkylates a cysteinyl residue of similar high reactivity. The substrate analogue, levulinic acid, a competitive inhibitor, partially protected the enzyme from inactivation by iodoacetate, suggesting that this alkylation was active site directed. In contrast, levulinic acid potentiates the inactivation caused by iodoacetamide. The product of the reaction, porphobilinogen, however, partially protected the enzyme from inactivation by both iodoacetate and iodoacetamide. Equilibrium dialysis studies of [Wllevulinic acid binding to the enzyme have shown that native enzyme (thiol-activated or air-oxidized) and the alkylated enzyme derivatives bind the same maximum number of moles of levulinic acid/ mol of enzyme (2/285,000 daltons). However, the binding affinities of the native thiol-activated enzyme and the iodoacetamide-inactivated enzyme were 3to 5-fold greater than those of the air-oxidized and iodoacetate-inactivated samples of enzyme. Peptide fingerprints, molecular sieve, and cation exchange chromatography of tryptic digests of the enzyme inactivated by iodo[Wlacetate or by iodo[Wlacetamide demonstrated that each reagent alkylated a cysteinyl residue which gave rise to a different radioactive tryptic peptide. Preliminarily, this suggests that each reagent alkyl-