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Jaffe EK , Petrovich RM , Volin M
5-Chlorolevulinate interactions with Bradyrhizobium japonicum porphobilinogen synthase
Bioorganic Chemistry. 1995 Dec;23(4) :450-459
PMID: ISI:A1995TP75300011   
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Abstract
5-Chlorolevulinic acid (5-CLA) is an alpha-chloroketone analog of the tetrapyrrole precursor 5-aminolevulinate (ALA). As such it serves as an excellent probe of the mechanisms of enzymes which form or utilize ALA. The enzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of ALA to form the monopyrrole porphobilinogen. In principle, 5-CLA could bind at either or both of the two different ALA binding sites of PBGS. In the presence of an active-site nucleophile, 5-CLA might chemically modify the protein with concomitant inactivation. Such inactivation has been shown for mammalian and Escherichia coli PBGS, each of which has reactive cysteines at the active site. In contrast, we report here that 5-CLA does not readily inactivate Bradyrhizobium japonicum PBGS (BjPBGS) in either the presence or the absence of ALA. At neutral pH 5- CLA is a reversible inhibitor of BjPBGS (K-i = 47 mu M) and affects both the K-m for ALA (0.8 mM) and the V-max of the reaction. BjPBGS does not use 5-CLA as an alternative substrate to form the 11-chloro analog of porphobilinogen in a mixed condensation with ALA. C-13 NMR was used to observe 5-chloro- [1,4-C-13]levulinate bound at the BjPBGS active site. The C-13 NMR chemical shifts indicate that 5-CLA preferentially binds to the first ALA binding site and undergoes formation of an analog of the first Schiff base intermediate which normally occurs in the PBGS-catalyzed reaction. These data contribute to a growing body of mechanistic work which supports the proposition that carbon-nitrogen bond formation precedes carbon-carbon bond formation in the PBGS-catalyzed biosynthesis of porphobilinogen. (C) 1995 Academic Press, Inc.
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Times Cited: 2 English Article TP753 BIOORG CHEM