Charge Stabilization and Entropy Reduction of Central Lysine Residues in Fructose-Bisphosphate Aldolase
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- 作者：Miguel St-Jean ; Casimir Blonski ; Jurgen Sygusch
- 刊名：Biochemistry
- 出版年：2009
- 出版时间：June 2, 2009
- 年：2009
- 卷：48
- 期：21
- 页码：4528-4537
- 全文大小：876K
- 年卷期：v.48,no.21(June 2, 2009)
- ISSN：1520-4995
文摘
Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon−carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34−65) flanking the active site as well as pKa reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pKa reduction and side chain disorder would further compromise charge stabilization during C−C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C−C bond cleavage and Schiff base−enamine interconversion.