Ramakrishna S, Benjamin W B
Biochem J. 1981 Jun 1;195(3):735-43. doi: 10.1042/bj1950735.
Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.
大鼠肝脏ATP柠檬酸裂解酶可被2,3 - 丁二酮和苯乙二醛灭活。在pH 8的4-（2-羟乙基）-1-哌嗪-乙磺酸缓冲液中，苯乙二醛能使酶活性最快且最完全地失活。丁二酮和苯乙二醛的灭活作用均呈浓度依赖性，并遵循准一级动力学。苯乙二醛还能降低ATP柠檬酸裂解酶的自磷酸化（催化性磷酸化）。苯乙二醛和丁二酮导致的酶失活是由于酶精氨酸残基的修饰：修饰后的酶无法与辅酶A - 琼脂糖结合。随着失活程度增加，V下降，但Km值未改变。底物辅酶A（CoASH）以及辅酶A加柠檬酸能显著保护该酶免于失活，但ATP提供的保护作用很小。用过量试剂进行失活处理时，每个酶单体约有8个精氨酸残基被修饰。柠檬酸、辅酶A和ATP能保护两到三个精氨酸残基不被苯乙二醛修饰。通过统计学方法对数据进行分析表明，失活是由于每个裂解酶单体中有一个必需的精氨酸残基被修饰，该残基被修饰的速度比其他精氨酸残基快1.5倍。我们的结果表明，这个必需的精氨酸残基位于辅酶A结合位点。
