Tovar-Méndez A, Mújica-Jiménez C, Muñoz-Clares R A
Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D.F., Mexico.
Biochim Biophys Acta. 1997 Feb 8;1337(2):207-16. doi: 10.1016/s0167-4838(96)00166-5.
Incubation of the nonphosphorylated form of maize-leaf phospho enol pyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5'-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7 +/- 0.3 s-1 M-1 and a first-order reverse rate constant of 0.0046 +/- 0.0002 s-1. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.
将玉米叶片磷酸烯醇式丙酮酸羧化酶（正磷酸：草酰乙酸羧基裂解酶（磷酸化），PEPC，EC 4.1.1.31）的非磷酸化形式与试剂磷酸吡哆醛（PLP）一起温育，会导致时间依赖性、可逆的失活以及对激活剂葡萄糖6-磷酸（Glc6P）和其他相关磷酸化化合物的脱敏。这两个过程并无关联，因为：（i）当在活性位点的饱和配体存在下进行PLP修饰（这可防止失活）时，仍可观察到对Glc6P的脱敏现象；（ii）在某些实验条件下，脱敏反应比失活反应快4倍。对Glc6P的脱敏反应对PLP呈一级反应，正向二级速率常数为4.7±0.3 s⁻¹ M⁻¹，逆向一级速率常数为0.0046±0.0002 s⁻¹。对剩余Glc6P敏感性与每摩尔酶亚基掺入的PLP残基摩尔数之间的相关性研究表明，酶单体的一个赖氨酰基团参与了酶对Glc6P的敏感性。聚乙二醇和甘油会增加该基团的反应性，而活性位点赖氨酰基团的反应性不受这些有机共溶质的影响。在存在但非不存在有机共溶质的情况下（有机共溶质），Glc6P自身可提供显著的抗脱敏保护作用，同时会增加失活程度。游离的PEP或PEP-Mg具有相反的作用，可保护酶免于失活并增加脱敏程度。它们还增强了Glc6P提供的抗脱敏保护作用。最后，PEPC抑制剂苹果酸对失活和脱敏均提供一定保护。综上所述，这些结果与变构Glc6P位点处或其附近一个高反应性赖氨酰基团的PLP修饰相一致。
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