Kiefer P M, Varughese K I, Su Y, Xuong N H, Chang C F, Gupta P, Bray T, Whiteley J M
University of California at San Diego, La Jolla, California 92093-0317, USA.
J Biol Chem. 1996 Feb 16;271(7):3437-44. doi: 10.1074/jbc.271.7.3437.
Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.
已构建了大鼠二氢蝶啶还原酶（EC 1.6.99.7）的9个单基因突变体，即D37I、W86I、Y146F、Y146H、K150Q、K150I、K150M、N186A和A133S，以及1个双基因突变体Y146F/K150Q。这些突变体在大肠杆菌中过表达，并对其蛋白质进行了纯化。其中，W86I、Y146F、Y146H、Y146F/K150Q和A133S这5个突变体已结晶并进行了结构表征。除N186A因过于不稳定而无法以均一形式分离外，已得出了每种突变酶形式的动力学常数，并且在有结构数据的5个实例中，通过与这些结构的关联解释了活性的改变。很明显，脱辅基酶与辅因子NADH的特异性相互作用对于整个蛋白质三级结构的完整性至关重要，并且活性位点的形成除了需要合适定位的W86外，还需要结合的辅因子。因此，当与辅因子形成氢键的三个主要中心发生突变，即37、150和186位时，会形成不稳定的部分活性酶。同样明显的是，酪氨酸146对酶的活性至关重要，因为Y146F突变体几乎无活性，仅具有野生型活性的1.1%。然而，当进行额外的K150Q突变时，Lys150附近水分子的重排伴随着50%野生型活性的恢复。有人认为水分子的参与补偿了酪氨酰羟基的损失。146位酪氨酸和组氨酸残基之间的差异体现在后者与底物形成的氢键几何结构相对不利，使活性降至野生型的15%。突变体A133S的活性几乎没有变化；然而，其羟基取代基通过提供一个可能的额外质子受体对活性位点有贡献。这对二氢蝶啶还原酶的价值不大，但在顺序类似的短链脱氢酶/还原酶中可能很重要，在这些酶中丝氨酸是该位置的首选氨基酸。
