Iglesias A A, Kakefuda G, Preiss J
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Protein Chem. 1992 Apr;11(2):119-28. doi: 10.1007/BF01025217.
ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacterium Synechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl2 enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form. Vmax at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.
通过用精氨酸试剂苯乙二醛处理，来自集胞藻PCC 6803的ADP葡萄糖焦磷酸化酶（EC 2.7.7.27）对变构配体的作用不敏感。当在3-磷酸甘油酸激活的条件下测定酶活性时，苯乙二醛对酶的修饰导致酶失活。在没有激活剂的情况下测定的催化活性几乎没有损失。无机磷酸（Pi）、3-磷酸甘油酸和磷酸吡哆醛能够保护该酶不被失活，而底物提供的保护作用最小。Pi和3-磷酸甘油酸表现出的保护作用取决于效应物浓度。氯化镁增强了3-磷酸甘油酸提供的保护作用。与未修饰形式相比，经苯乙二醛部分修饰的酶对3-磷酸甘油酸激活和Pi抑制更具抗性。在饱和3-磷酸甘油酸浓度下的最大反应速度（Vmax）以及酶对Pi的表观亲和力在苯乙二醛修饰后降低。标记的苯乙二醛掺入酶中的量与活性损失成正比。Pi和3-磷酸甘油酸几乎完全阻止了该试剂掺入蛋白质中。结果表明，每摩尔酶亚基中有一个精氨酸残基参与蓝细菌ADP葡萄糖焦磷酸化酶中变构效应物的结合。
