Department of Chemistry and Biochemistry, University of Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA.
Arch Biochem Biophys. 2011 Sep 15;513(2):71-80. doi: 10.1016/j.abb.2011.07.009. Epub 2011 Jul 28.
Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E(t) and V₁/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.
氨甲酰磷酸脱氢酶催化 NAD 依赖性的反应将氨甲酰磷酸转化为 L-赖氨酸和α-酮戊二酸。在apo 酶的结构中，观察到二核苷酸结合位点附近的半胱氨酸 205 和半胱氨酸 249 之间存在一个二硫键，而在结合 AMP 的结构中观察到一个二硫键，表明二核苷酸优先与还原酶结合。将 C205 突变为 S 会增加 C205S 在 pH 7 时的 V/E(t) 和 V/KE(t) 值。初级氘和溶剂氘动力学同位素效应表明，包括氢化物转移和水解步骤的催化途径在 C205S 中对限速的贡献更大，但这两个步骤相对于彼此的速率保持不变。与 WT 相比，在 pH 值低于 7 时，V₁/E(t)和 V₁/K(NAD)Et 的速率常数会大大增加。数据表明，活性的低 pH 值增加是由于 C205S 突变酶对 pK(a)约为 7 的酶基团质子化状态的敏感性降低所致，这可能导致 pH 依赖性构象变化。用 TCEP 还原 WT 和 C205S 突变酶在 pH 6 时给出相等的活性，与观察到的 C205S 突变酶的活性增加一致。
