Dhalla A M, Li B, Alibhai M F, Yost K J, Hemmingsen J M, Atkins W M, Schineller J, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Protein Sci. 1994 Mar;3(3):476-81. doi: 10.1002/pro.5560030313.
In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)
为了了解ATP和L-谷氨酸与谷氨酰胺合成酶的结合性质,以及精氨酸339和精氨酸359在催化过程中的作用,通过定点诱变将这些氨基酸替换为半胱氨酸。在339位和359位的单个突变(精氨酸→半胱氨酸)导致催化活性急剧下降。此外,底物ATP和谷氨酸的米氏常数(Km值)大幅高于野生型(WT)酶的值。依次将每个半胱氨酸化学修饰为精氨酸“类似物”,试图通过共价修饰“挽救”催化活性;2-氯乙脒(CA)(产生硫醚)和2,2'-二硫代双(乙脒)(DTBA)(产生二硫键)是用于实现这些化学转化的试剂。与CA反应后,R339C和R359C突变体均显示催化活性显著恢复(分别为WT的50%和70%),且ATP的Km值降至接近WT酶的值。用DTBA处理时,化学修饰的R339C的催化常数(kcat)高于WT谷氨酰胺合成酶,但化学修饰的R359C仅恢复了少量活性。DTBA对每个突变体的修饰是定量的,且每个修饰后的酶对ATP和谷氨酸的Km值相似。如预期含有二硫键的修饰酶一样,用二硫苏糖醇处理可使DTBA修饰的R339C的高催化活性恢复到未修饰的R339C的水平。使用3-溴丙胺(BPA)将每个含半胱氨酸的突变体修饰为赖氨酸“类似物”。(摘要截断于250字)