Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme.
Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme.
复制标题
酿酒酵母高异柠檬酸脱氢酶催化机制中诱导构象变化的证据：D271N 突变酶的表征。
DOI：
10.1016/j.abb.2015.08.016
复制
发表时间：
2015
影响因子：
3.9
通讯作者：
Cook,PaulF
中科院分区：
文献类型：
--
作者：
Hsu,Chaonan;West,AnnH;Cook,PaulF
Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD+-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for thede novosynthesis ofl-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys–Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of theSaccharomyces cerevisiaeHIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008)Biochemistry47, 4169–4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized using initial rate studies. A decrease of 520-fold was observed inVandV/KMgHIc, suggesting the same step(s) limit the reaction at limiting and saturating MgHIc concentrations. Solvent kinetic deuterium isotope effects (SKIE) and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations. In addition, at limiting MgHIc, an inverse (SKIE) of 0.7 coupled to a significant normal effect of viscosogen (2.1) indicates equilibrium binding of MgHIc prior to the rate-limiting conformational change. The maximum rate exhibits a small partial change at high pH suggesting a pH-dependent conformational change, whileV/KMgHIcexhibits the same partial change observed in V, and a decrease at low pH with a pKaof 6 reflecting the requirement for the unprotonated form of MgHIc to bind to enzyme. However, neither parameter reflects the pH dependence of the chemical reaction. This pH independence of the chemical reaction over the range 5.5–9.5 is consistent with the much slower conformational change that would effectively perturb the observed pKvalues for catalytic groups to lower and higher pH. In other words, the pH dependence of the chemical reaction will only be observed when chemistry becomes slower than the rate of the conformational change. Data support the hypothesis of the existence of a pre-catalytic conformational change coupled to the binding of MgHIc.
