Hydroxynaphthaldehyde Phosphate Derivatives as Potent Covalent Schiff Base Inhibitors of Fructose-1,6-bisphosphate Aldolase

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• 作者：Chantal Dax ; Mathieu Coinç ; on ; Jurgen Sygusch ; and Casimir Blonski
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：April 12, 2005
• 年：2005
• 卷：44
• 期：14
• 页码：5430 - 5443
• 全文大小：362K
• 年卷期：v.44,no.14(April 12, 2005)
• ISSN：1520-4995

文摘

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P₂) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbitmuscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, massspectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P₂, whereasHNA-P exhibited slow-binding inhibition with an overall inhibition constant of ~24 nM. HNA-Pinactivation was very slowly reversed with t_1/2 ~10 days. Mass spectrometry and spectrophotometricabsorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivationand Schiff base formation by HNA-P were identical and corresponded to ~4 HNA-P molecules boundpar aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysineresidues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK_a~8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, anactive site residue that binds the C₁-phosphate of dihydroxyacetone phosphate, diminished HNA-P bindingand enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphateinteractions with the C₁-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl,ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation byintramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitiveinhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significantconformational barrier to bond formation as well as electrostatic destabilization of protonated ketimineintermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, andin those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysiswas not mechanistically favorable. The findings at the molecular level corroborate the observed mechanismof slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for futurealdolase inhibitor design.