Hartman F C, Milanez S, Lee E H
J Biol Chem. 1985 Nov 15;260(26):13968-75.
Trinitrobenzene sulfonate rapidly inactivates ribulosebisphosphate carboxylase/oxygenase from both spinach and Rhodospirillum rubrum. With large molar excesses of the reagent, the reactions obey pseudo-first order kinetics and the rates of inactivations are directly proportional to the concentrations of trinitrobenzene sulfonate; thus, there is no indication of reversible complexation of reagent with enzyme. Saturating levels of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduce the rates of inactivations but do not prevent them, thereby suggesting that the groups subject to arylation remain accessible in the enzyme complexed with competitive inhibitor. Characterization of tryptic digests of the inactivated enzymes reveals that Lys-166 of the R. rubrum enzyme and Lys-334 of the spinach enzyme are the only major sites of arylation. Both of these lysines have been assigned to the catalytic site by prior affinity labeling studies and are found within highly conserved regions of primary structure. As a monoanion over a wide pH range, trinitrobenzene sulfonate, for which the carboxylase lacks high affinity, can thus be used to determine the pKa values of the two active-site lysyl epsilon-amino groups. Based on the pH dependency of inactivation of the R. rubrum enzyme by trinitrobenzene sulfonate, the epsilon-amino group of Lys-166 exhibits a pKa of 7.9 and an intrinsic reactivity (ko) of 670 M-1 min-1. In analogous experiments, Lys-334 of the spinach enzyme exhibits a pKa of 9.0 and a ko of 4500 M-1 min-1. Under deactivation conditions (i.e. in the absence of CO2 and Mg2+), the pKa of Lys-334 becomes 9.8 and the ko is increased to 26,000 M-1 min-1. By comparison, the reaction of trinitrobenzene sulfonate with N-alpha-acetyl-lysine reveals a pKa of 10.8 and a ko of 1250 M-1 min-1. The spinach carboxylase, catalytically inactive as a consequence of selective arylation of Lys-334, still exhibits tight binding of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate. Therefore, Lys-334 is not required for substrate binding and may serve a role in catalysis. The unusually low pKa of Lys-166 argues that this residue is also important to catalysis rather than substrate binding.
三硝基苯磺酸盐能迅速使菠菜和深红红螺菌的核酮糖-1,5-二磷酸羧化酶/加氧酶失活。当试剂的摩尔过量很大时，反应遵循准一级动力学，失活速率与三硝基苯磺酸盐的浓度成正比；因此，没有迹象表明试剂与酶发生可逆络合。竞争性抑制剂2-羧基核糖醇1,5-二磷酸的饱和水平会降低失活速率，但不能阻止失活，这表明与竞争性抑制剂结合的酶中易于被芳基化的基团仍然可及。对失活酶的胰蛋白酶消化产物的表征表明，深红红螺菌酶的Lys-166和菠菜酶的Lys-334是唯一主要的芳基化位点。通过先前的亲和标记研究，这两个赖氨酸都已被定位到催化位点，并且在一级结构的高度保守区域中被发现。作为在很宽的pH范围内的单阴离子，羧化酶对其缺乏高亲和力的三硝基苯磺酸盐，因此可用于确定两个活性位点赖氨酰ε-氨基的pKa值。基于三硝基苯磺酸盐对深红红螺菌酶失活的pH依赖性，Lys-166的ε-氨基的pKa为7.9，固有反应性(ko)为670 M-1 min-1。在类似的实验中，菠菜酶的Lys-334的pKa为9.0，ko为4500 M-1 min-1。在失活条件下（即不存在CO2和Mg2+时），Lys-334的pKa变为9.8，ko增加到26,000 M-1 min-1。相比之下，三硝基苯磺酸盐与N-α-乙酰赖氨酸的反应的pKa为10.8，ko为1250 M-1 min-1。由于Lys-334的选择性芳基化而催化失活的菠菜羧化酶，仍然表现出对过渡态类似物2-羧基阿拉伯糖醇1,5-二磷酸的紧密结合。因此，底物结合不需要Lys-334，它可能在催化中起作用。Lys-166异常低的pKa表明该残基对催化而不是底物结合也很重要。
