Relyea Heather A, Vrtis Jennifer M, Woodyer Ryan, Rimkus Stacey A, van der Donk Wilfred A
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Biochemistry. 2005 May 3;44(17):6640-9. doi: 10.1021/bi047640p.
Phosphite dehydrogenase (PTDH) catalyzes the NAD-dependent oxidation of phosphite to phosphate, a reaction that is 15 kcal/mol exergonic. The enzyme belongs to the family of D-hydroxy acid dehydrogenases. Five other family members that were analyzed do not catalyze the oxidation of phosphite, ruling out the possibility that this is a ubiquitous activity of these proteins. PTDH does not accept any alternative substrates such as thiophosphite, hydrated aldehydes, and methylphosphinate, and potential small nucleophiles such as hydroxylamine, fluoride, methanol, and trifluoromethanol do not compete with water in the displacement of the hydride from phosphite. The pH dependence of k(cat)/K(m,phosphite) is bell-shaped with a pK(a) of 6.8 for the acidic limb and a pK(a) of 7.8 for the basic limb. The pK(a) of 6.8 is assigned to the second deprotonation of phosphite. However, whether the dianionic form of phosphite is the true substrate is not clear since a reverse protonation mechanism is also consistent with the available data. Unlike k(cat)/K(m,phosphite), k(cat) and k(cat)/K(m,NAD) are pH-independent. Sulfite is a strong inhibitor of PTDH that is competitive with respect to phosphite and uncompetitive with respect to NAD(+). Incubation of the enzyme with NAD(+) and low concentrations of sulfite results in a covalent adduct between NAD(+) and sulfite in the active site of the enzyme that binds very tightly. Fluorescent titration studies provided the apparent dissociation constants for NAD(+), NADH, sulfite, and the sulfite-NAD(+) adduct. Substrate isotope effect studies with deuterium-labeled phosphite resulted in small normal isotope effects (1.4-2.1) on both k(cat) and k(cat)/K(m,phosphite) at pH 7.25 and 8.0. Solvent isotope effects (SIEs) on k(cat) are similar in size; however, the SIE of k(cat)/K(m,phosphite) at pH 7.25 is significantly larger (4.4), whereas at pH 8.0, it is the inverse (0.6). The pH-rate profile of k(cat)/K(m,phosphite), which predicts that the observed SIEs will have a significant thermodynamic origin, can account for these effects.
亚磷酸脱氢酶（PTDH）催化亚磷酸依赖NAD氧化为磷酸，该反应放能15千卡/摩尔。该酶属于D-羟基酸脱氢酶家族。分析的其他五个家族成员不催化亚磷酸的氧化，排除了这是这些蛋白质普遍存在的活性的可能性。PTDH不接受任何替代底物，如硫代亚磷酸酯、水合醛和甲基次膦酸酯，并且潜在的小亲核试剂，如羟胺、氟化物、甲醇和三氟甲醇，在从亚磷酸中取代氢化物时不与水竞争。k(cat)/K(m,亚磷酸)对pH的依赖性呈钟形，酸性部分的pK(a)为6.8，碱性部分的pK(a)为7.8。6.8的pK(a)归因于亚磷酸的第二次去质子化。然而，亚磷酸的二阴离子形式是否是真正的底物尚不清楚，因为反向质子化机制也与现有数据一致。与k(cat)/K(m,亚磷酸)不同，k(cat)和k(cat)/K(m,NAD)与pH无关。亚硫酸盐是PTDH的强抑制剂，对亚磷酸具有竞争性，对NAD(+)具有非竞争性。将该酶与NAD(+)和低浓度亚硫酸盐一起孵育会导致NAD(+)与亚硫酸盐在酶的活性位点形成紧密结合的共价加合物。荧光滴定研究提供了NAD(+)、NADH、亚硫酸盐和亚硫酸盐-NAD(+)加合物的表观解离常数。用氘标记的亚磷酸进行的底物同位素效应研究在pH 7.25和8.0时对k(cat)和k(cat)/K(m,亚磷酸)都产生了较小的正常同位素效应（1.4 - 2.1）。对k(cat)的溶剂同位素效应（SIEs）大小相似；然而，在pH 7.25时k(cat)/K(m,亚磷酸)的SIE明显更大（4.4），而在pH 8.0时则相反（0.6）。k(cat)/K(m,亚磷酸)的pH - 速率曲线预测观察到的SIEs将有显著的热力学起源，可以解释这些效应。
