Inhibition Studies of Porphobilinogen Synthase fromEscherichia coli Differentiating between the Two Recognition Sites

Stauffer, F.; Zizzari, Eleonora; Engeloch-Jarret, Caroline; Faurite, Jean-Philippe; Bobáľová, Janette; Neier, Reinhard

doi:10.1002/1439-7633(20010504)2:5<343::aid-cbic343>3.0.co;2-1

Cited by 10 publications

(7 citation statements)

References 55 publications

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“…If C–C bond formation is first, the intermediate will take the form of a seven carbon chain diacid whilst if C–N bond formation occurs first, a 10 atom chain diacid will be formed [20–22]. As has been noted previously, dicarboxylic acids may be able to cross‐link the carboxylic acid binding groups associated with the A and P substrate binding sites [23,24]. Because of this potential we have crystallised the ALAD from Saccharomyces cerevisiae in complex with the 10 carbon chain diacid irreversible inhibitors 4‐oxosebacic acid and 4,7‐dioxosebacic acid (formulae are shown in Fig.…”

Section: Introductionmentioning

confidence: 94%

The X‐ray structure of yeast 5‐aminolaevulinic acid dehydratase complexed with two diacid inhibitors

et al. 2001

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The structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A-and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A-and P-sites is proposed. ß

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Section: Introductionmentioning

confidence: 94%

The X‐ray structure of yeast 5‐aminolaevulinic acid dehydratase complexed with two diacid inhibitors

et al. 2001

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“…1A). Despite the fact that much is known about the PBGS structure, the sequence of bond-making and bond-breaking events that follow formation of the ternary complex of PBGS with the two substrate molecules is the subject of active discussion (2)(3)(4)(5). Based on an extensive phylogenetic variation in the number and kinds of metal ions used for catalytic and/or allosteric roles, it is possible that the order of chemical events in the enzymecatalyzed reaction mechanism may not be phylogenetically conserved.…”

mentioning

confidence: 99%

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic Acid

Jaffe¹,

Kervinen²,

Martins³

et al. 2002

Journal of Biological Chemistry

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Porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA), an essential step in tetrapyrrole biosynthesis. 4-Oxosebacic acid (4-OSA) and 4,7-dioxosebacic acid (4,7-DOSA) are bisubstrate reaction intermediate analogs for PBGS. We show that 4-OSA is an active sitedirected irreversible inhibitor for Escherichia coli PBGS, whereas human, pea, Pseudomonas aeruginosa, and Bradyrhizobium japonicum PBGS are insensitive to inhibition by 4-OSA. Some variants of human PBGS (engineered to resemble E. coli PBGS) have increased sensitivity to inactivation by 4-OSA, suggesting a structural basis for the specificity. The specificity of 4-OSA as a PBGS inhibitor is significantly narrower than that of 4,7-DOSA. Comparison of the crystal structures for E. coli PBGS inactivated by 4-OSA versus 4,7-DOSA shows significant variation in the half of the inhibitor that mimics the second substrate molecule (A-side ALA). Compensatory changes occur in the structure of the active site lid, which suggests that similar changes normally occur to accommodate numerous hybridization changes that must occur at C3 of A-side ALA during the PBGS-catalyzed reaction. A comparison of these with other PBGS structures identifies highly conserved active site water molecules, which are isolated from bulk solvent and implicated as proton acceptors in the PBGScatalyzed reaction.

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“…Both inhibitors are like ALA in that they bind to a conserved lysine at one of the two active sites, known as the P-site, as visualized by crystal structure analysis (Erskine et al 2001). As proposed by others (Stauffer et al 2001), levulinic acid competes with ALA for binding to the second site, the A-site, resulting in the competitive behaviour observed with the Pieris enzyme. These authors further propose that in the case of uncompetitive or mixed inhibition, as was the case with succinylacetone in the present study, the inhibitor also competes at the P-site or at both sites.…”

Section: Discussionmentioning

confidence: 67%

“…The Lineweaver-Burk plot for PBGS from Pieris was linear, demonstrating normal Michaelis-Menten kinetics under our experimental conditions (cf., Stauffer et al 2001). The specific activity of ∼280 µmol h -1 mg -1 at 50°C corresponds to ∼160 µmol h -1 mg -1 at 37°C at which PBGS enzymes are commonly studied.…”

Section: Discussionmentioning

confidence: 99%

Porphobilinogen Synthase from the Butterfly,Pieris brassicae: Purification and Comparative Characterization

Gessel

Kayser

2007

Journal of Insect Science

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Porphobilinogen represents a key building block of tetrapyrroles serving as functional ligands of many vitally important proteins. Here we report the first purification of porphobilinogen synthase (PBGS) from whole insects by sequentially employing two modes of native electrophoresis on polyacrylamide gels subsequent to more conventional procedures. Using adults of Pieris brassicae L. (Lepidoptera: Pieridae) we achieved ∼10,000-fold purification with final yields of up to 25% of electrophoretically pure PBGS with a specific activity of ∼160 µmol PBG h-1 mg-1 at 37°C and an affinity of 0.36 mM to its substrate 5-aminolevulinic acid. Enzyme activity was inhibited by the substrate mimics, levulinic acid and succinylacetone, and by chelating agents. PBGS behaved as a relatively heat-stable octameric complex of 292.3 kDa composed of 36.5 kDa subunits. Most general features of this insect PBGS were comparable to those published for other animal PBGS enzymes, while remarkable differences were found to the reported recombinant Drosophila enzyme. Moreover, rabbit antiserum directed against purified Pieris PBGS revealed significant immunological differences among insect PBGS enzymes from a wide range of orders contrasting to the overall evolutionary conserved features of this enzyme.

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Inhibition Studies of Porphobilinogen Synthase fromEscherichia coli Differentiating between the Two Recognition Sites

Cited by 10 publications

References 55 publications

The X‐ray structure of yeast 5‐aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The X‐ray structure of yeast 5‐aminolaevulinic acid dehydratase complexed with two diacid inhibitors

Species-specific Inhibition of Porphobilinogen Synthase by 4-Oxosebacic Acid

Porphobilinogen Synthase from the Butterfly,Pieris brassicae: Purification and Comparative Characterization

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