Cheng K C, Nowak T
Department of Chemistry, University of Notre Dame, Indiana 46556.
J Biol Chem. 1989 Feb 25;264(6):3317-24.
The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.
通过化学修饰并对修饰后的酶进行表征，研究了精氨酸在禽肝磷酸烯醇丙酮酸羧激酶活性位点的存在情况。精氨酸特异性试剂苯乙二醛、2,3-丁二酮和1,2-环己二酮均不可逆地抑制该酶，二级速率常数分别为3.42 M-1 min-1、3.13 M-1 min-1和0.313 M-1 min-1。底物磷酸烯醇丙酮酸、IDP和激活剂Mn2+对抑制作用的保护作用很小或适中。单独的CO2或在任何其他底物存在下的CO2都能有效防止修饰。CO2对苯乙二醛或1,2-环己二酮修饰的保护呈现双相模式。活性迅速丧失至40%-60%，随后非常缓慢地丧失。抑制动力学表明精氨酸的修饰是特异性的，并导致酶活性丧失。底物保护研究表明，磷酸烯醇丙酮酸羧激酶的CO2位点存在一个或多个精氨酸残基。显然，含磷酸底物的结合位点没有精氨酸残基。在CO2存在下形成的部分无活性（活性为40%-60%）的酶，其动力学常数略有变化，动力学、质子弛豫速率和圆二色性研究表明其结合参数和二级结构没有改变。在CO2存在下（活性为40%-60%）用[(7-)14C]苯乙二醛标记酶，结果显示每摩尔酶有2摩尔苯乙二醛修饰，即1个精氨酸或半胱氨酸残基被修饰。在没有CO2的情况下对磷酸烯醇丙酮酸羧激酶进行标记，每摩尔酶产生6摩尔标记。标记结果表明，禽磷酸烯醇丙酮酸羧激酶在总共52个氨基酸残基中有2个或3个反应性精氨酸残基，其中只有1个或2个位于活性位点，参与CO2的结合和激活。
