Hydroxynaphthaldehyde Phosphate Derivatives as Potent Covalent Schiff Base Inhibitors of Fructose-1,6-bisphosphate Aldolase
| Autor: | Mathieu Coinçon, Jurgen Sygusch, Casimir Blonski, Chantal Dax |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Magnetic Resonance Spectroscopy
Schiff base biology Stereochemistry Aldolase A Active site Naphthols Biochemistry Organophosphates Kinetics chemistry.chemical_compound Non-competitive inhibition chemistry Covalent bond Fructose-Bisphosphate Aldolase Mutagenesis Site-Directed biology.protein Animals Rabbits Enzyme kinetics Enzyme Inhibitors Binding site Muscle Skeletal Schiff Bases Dihydroxyacetone phosphate |
| Zdroj: | Biochemistry. 44:5430-5443 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi0477992 |
| Popis: | Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design. |
| Databáze: | OpenAIRE |
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