The schiff base complex of yeast 5‐aminolaevulinic acid dehydratase with laevulinic acid

Abstract

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase~ALAD! and the inhibitor laevulinic acid has been determined at 2.15 Å resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain ϾNH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines~133, 135, and 143!. Inhibitor binding caused a substantial ordering of the active site flap~residues 217-235!, which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

“…Thus, Schiff base formation with ALA is not responsible for a conformational change that dictates half-site reactivity. This is consistent with crystallographic data that show Schiff base formation to levulinic acid does not cause protein asymmetry (7,9). It remains possible that binding of the C-5 amino group of P-side ALA, which is the ALA that forms the Schiff base, is essential for the evolution of the asymmetric structure.…”

“…By analogy to wild type, we assume that the more upfield signal (127-129 ppm range) arises from C-5 and the more downfield signal (121-124 ppm range) arises from C-3. In all cases the C-5 signal of enzyme-bound product is 6.3-7.7 ppm upfield from the chemical shift of free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen. From this we conclude that the active site lysine is not a major contributor to the massive shift seen at C-5.…”

“…However, since to date 13 C-labeled porphobilinogen bound to all Zn(II)-requiring PBGS has shown identical chemical shifts (2,12), 13 C-labeled porphobilinogen bound to K252G is not expected to provide different information than that available from the E. coli PBGS variant K246G. In the case of K246W, only free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen (and a degradation product of unknown structure) was observed at both pH 7 and pH 8. The line widths of free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen and the degradation product were quite sharp (ϳ7 Hz) confirming their unbound state.…”

“…In the case of K246W, only free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen (and a degradation product of unknown structure) was observed at both pH 7 and pH 8. The line widths of free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen and the degradation product were quite sharp (ϳ7 Hz) confirming their unbound state. We conclude that the presence of tryptophan prevents porphobilinogen binding to the active site due to steric hindrance.…”

“…For wild-type E. coli PBGS, the control samples, which included all the physical manipulations to the protein, retained complete catalytic activity. In contrast, when treated with both [4][5][6][7][8][9][10][11][12][13][14] C]ALA and NaBH 4 , three samples of wild-type PBGS each showed ϳ80% inactivation and 14 C labeling at 0.42-0.49 ALA/subunit. This stoichiometry is consistent with four functional active sites per octamer.…”