Kurland I, Chapman B, Lee Y H, Pilkis S
Division of Endocrinology and Metabolism UCLA School of Medicine 90024-1736, USA.
Biochem Biophys Res Commun. 1995 Aug 15;213(2):663-72. doi: 10.1006/bbrc.1995.2183.
Arg-104 of the kinase domain of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was mutated to alanine, the mutant enzyme expressed in E. coli with a T7 RNA polymerase-based expression system, and purified to homogeneity by Blue-Sepharose and Q-Sepharose chromatography. The mutant enzyme exhibited a 200-fold increase in Km for fructose-6-phosphate, no change in Km for ATP, and a 2-3-fold increase in catalytic rate. The results indicate that Arg-104, along with Arg-195, are the principal binding site residues for the 6-phosphate group of fructose-6-phosphate. In contrast to the corresponding residue in the related E. coli 6-phosphofructo-1-kinase, Arg-104 did not stabilize the transition state at pH 7-9. The Arg-104 mutation also decreased Fru-2,6-P2ase activity without affecting substrate inhibition, which suggests that this mutation affects the bisphosphatase active site conformation and/or substrate access to it.
将6-磷酸果糖-2-激酶/果糖2,6-二磷酸酶激酶结构域的第104位精氨酸突变为丙氨酸，利用基于T7 RNA聚合酶的表达系统在大肠杆菌中表达突变酶，并通过蓝琼脂糖凝胶和Q琼脂糖凝胶色谱法纯化至同质。突变酶对6-磷酸果糖的Km值增加了200倍，对ATP的Km值没有变化，催化速率增加了2至3倍。结果表明，精氨酸104与精氨酸195一起，是6-磷酸果糖6-磷酸基团的主要结合位点残基。与相关的大肠杆菌6-磷酸果糖-1-激酶中的相应残基不同，精氨酸104在pH 7至9时不稳定过渡态。精氨酸104突变也降低了果糖-2,6-二磷酸酶活性，而不影响底物抑制，这表明该突变影响双磷酸酶活性位点构象和/或底物对其的可及性。
