Valentini G, Iadarola P, Ferri G, Speranza M L
Dipartimento di Biochimica, Università di Pavia, Italy.
Biol Chem Hoppe Seyler. 1995 Apr;376(4):231-5. doi: 10.1515/bchm3.1995.376.4.231.
The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.
来自大肠杆菌的变构调节型丙酮酸激酶I（PKI）被ATP类似物2'，3'-二醛ATP（o-ATP）灭活，其抑制常数（Ki）为3.6 mM。ATP和磷酸烯醇丙酮酸可保护该酶的活性，而异构激活剂1,6-二磷酸果糖则提高了灭活速率。用o-ATP孵育，然后用放射性硼氢化钠还原形成的席夫碱，用于确定PKI的ATP结合位点。经胰蛋白酶消化、标记肽段纯化及序列分析后，在PKI已知的DNA推导序列中鉴定出四个修饰的赖氨酸残基，即Lys173、Lys175、Lys272和Lys317。相邻的赖氨酸173和175以互斥方式与o-ATP反应，二者共占回收放射性的53%，其余分布在Lys272（31%）和Lys317（16%）上。当将这些修饰赖氨酸的位置与肌肉丙酮酸激酶现有的三维结构进行拟合时，可确定PKI上的催化性和变构性ATP结合位点。
