Reczkowski R S, Taylor J C, Markham G D
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Biochemistry. 1998 Sep 29;37(39):13499-506. doi: 10.1021/bi9811011.
S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.
S-腺苷甲硫氨酸（AdoMet）合成酶催化由ATP和L-甲硫氨酸形成AdoMet和三聚磷酸（PPPi），并在产物释放前将PPPi随后水解为PPi和Pi。对于在多磷酸链两端发生的两个连续反应的催化过程中涉及的活性位点残基的作用，人们了解甚少。大肠杆菌AdoMet合成酶的晶体学研究表明，精氨酸-244是多磷酸结合位点附近唯一的精氨酸。精氨酸-244作为第七个残基嵌入保守序列DxGxTxxKxI中，该序列也存在于无机焦磷酸酶的活性位点，提示存在一个潜在的焦磷酸结合基序。用精氨酸特异性试剂苯乙二醛或对羟基苯乙二醛对AdoMet合成酶进行化学修饰会使该酶失活。ATP和PPPi可保护该酶不被失活，这与在多磷酸结合位点附近存在一个重要的精氨酸残基一致。已利用定点诱变将保守的精氨酸-244替换为亮氨酸（R244L）或组氨酸（R244H）。在整个反应中，R244L突变体的催化常数（kcat）降低了约10³倍，底物米氏常数（Km）值增加了7至10倍；R244H突变体的kcat降低了约10⁵倍。相比之下，R244L和R244H突变体对添加的PPPi的水解kcat值降低不到2个数量级。与野生型酶不同，野生型酶中形成的98%的Pi源自ATP的γ-磷酸基团，而在R244L突变体中，PPPi中间体在活性位点的取向达到平衡，来自ATP的α-和γ-磷酸基团产生等量的Pi。因此，活性位点精氨酸在AdoMet形成过程中从ATP上切割PPPi以及在维持PPPi在活性位点的取向方面具有深远作用，而在随后的PPPi水解反应中作用较小。
