He Rui, Reyes Archie C, Amyes Tina L, Richard John P
Department of Chemistry , University at Buffalo, SUNY , Buffalo , New York 14260-3000 , United States.
Biochemistry. 2018 Jun 12;57(23):3227-3236. doi: 10.1021/acs.biochem.7b01282. Epub 2018 Feb 5.
The side chain of Q295 of glycerol-3-phosphate dehydrogenase from human liver ( hlGPDH) lies in a flexible loop, that folds over the phosphodianion of substrate dihydroxyacetone phosphate (DHAP). Q295 interacts with the side-chain cation from R269, which is ion-paired to the substrate phosphodianion. Kinetic parameters k/ K (M s) and k/ K K (M s) were determined, respectively, for catalysis of the reduction of DHAP and for dianion activation of catalysis of reduction of glycolaldehyde (GA) catalyzed by wild-type, Q295G, Q295S, Q295A, and Q295N mutants of hlGPDH. These mutations result in up to a 150-fold decrease in ( k/ K) and up to a 2.7 kcal/mol decrease in the intrinsic phosphodianion binding energy. The data define a linear correlation with slope 1.1, between the intrinsic phosphodianion binding energy and the intrinsic phosphite dianion binding energy for activation of hlGPDH-catalyzed reduction of GA, that demonstrates a role for Q295 in optimizing this dianion binding energy. The R269A mutation of wild-type GPDH results in a 9.1 kcal/mol destabilization of the transition state for reduction of DHAP, but the same R269A mutation of N270A and Q295A mutants result in smaller 5.9 and 4.9 kcal/mol transition-state destabilization. Similarly, the N270A or Q295A mutations of R269A GPDH each result in large falloffs in the efficiency of rescue of the R269A mutant by guanidine cation. We conclude that N270, which interacts for the substrate phosphodianion and Q295, which interacts with the guanidine side chain of R269, function to optimize the apparent transition-state stabilization provided by the cationic side chain of R269.
人肝脏甘油 - 3 - 磷酸脱氢酶（hlGPDH）的Q295侧链位于一个柔性环中，该环折叠在底物磷酸二羟丙酮（DHAP）的磷酸二阴离子上方。Q295与R269的侧链阳离子相互作用，R269与底物磷酸二阴离子形成离子对。分别测定了野生型、Q295G、Q295S、Q295A和Q295N突变体的hlGPDH催化DHAP还原以及二醇醛（GA）还原催化过程中磷酸二阴离子活化的动力学参数k/K（M⁻¹s⁻¹）和k/KK（M⁻²s⁻¹）。这些突变导致（k/K）下降高达150倍，内在磷酸二阴离子结合能下降高达2.7千卡/摩尔。数据定义了hlGPDH催化GA还原活化的内在磷酸二阴离子结合能与内在亚磷酸二阴离子结合能之间斜率为1.1的线性相关性，这表明Q295在优化这种二阴离子结合能中起作用。野生型GPDH的R269A突变导致DHAP还原的过渡态不稳定9.1千卡/摩尔，但N270A和Q295A突变体的相同R269A突变导致较小的5.9和4.9千卡/摩尔过渡态不稳定。同样，R269A GPDH的N270A或Q295A突变各自导致胍阳离子对R269A突变体的拯救效率大幅下降。我们得出结论，与底物磷酸二阴离子相互作用的N270和与R269的胍侧链相互作用的Q295，其作用是优化由R269阳离子侧链提供的表观过渡态稳定性。
