Bhagwat A S, McFadden B A
Arch Biochem Biophys. 1983 Jun;223(2):610-7. doi: 10.1016/0003-9861(83)90625-2.
Ribulose bisphosphate (RuBP) carboxylase is rapidly and irreversibly inactivated by photooxidation sensitized by pyridoxal phosphate. Both pyridoxal and pyridoxamine phosphate were much less effective in sensitizing the photooxidation even when used at twice the concentration of pyridoxal phosphate. These results imply that pyridoxal phosphate binds at the active site not only through a Schiff base, but also through ionic interaction with the phosphate binding region. Spectral analysis of the photooxidized enzyme showed a new absorption maximum at 325 nm due to reduction of the Schiff base between pyridoxal phosphate and a lysyl residue with concomitant oxidation of a histidine residue. The stoichiometry of photooxidative [3H]pyridoxal phosphate incorporation was 0.87 mol/mol of a 70,000-dalton large subunit-small subunit combination. Studies with 3H-labeled diethyl pyrocarbonate showed that both photooxidation and carbethoxylation occur at the same histidine residue. However, photooxidation by pyridoxal phosphate is very specific for an active site histidine residue due to the high specificity of this affinity label. Several competitive inhibitors with respect to ribulose bisphosphate offered appreciable protection against pyridoxal phosphate-induced photooxidation of the enzyme. The photooxidized enzyme showed an increase in the net negative charge on the protein which was evident from the higher mobility of the photooxidized enzyme toward the anode in polyacrylamide gel electrophoresis.
核酮糖二磷酸羧化酶会被磷酸吡哆醛敏化的光氧化作用迅速且不可逆地失活。即使磷酸吡哆醛和磷酸吡哆胺的使用浓度是磷酸吡哆醛的两倍，它们在敏化光氧化方面的效果也要差得多。这些结果表明，磷酸吡哆醛不仅通过席夫碱在活性位点结合，还通过与磷酸结合区域的离子相互作用结合。对光氧化酶的光谱分析表明，由于磷酸吡哆醛与赖氨酰残基之间的席夫碱还原，同时组氨酸残基氧化，在325nm处出现了一个新的吸收最大值。光氧化[³H]磷酸吡哆醛掺入的化学计量比为每摩尔70,000道尔顿的大亚基 - 小亚基组合0.87摩尔。用³H标记的焦碳酸二乙酯进行的研究表明，光氧化和乙氧羰基化都发生在同一个组氨酸残基上。然而，由于这种亲和标记的高特异性，磷酸吡哆醛的光氧化对活性位点组氨酸残基非常特异。几种相对于核酮糖二磷酸的竞争性抑制剂对磷酸吡哆醛诱导的酶光氧化提供了相当程度的保护。光氧化酶在蛋白质上的净负电荷增加，这在聚丙烯酰胺凝胶电泳中光氧化酶向阳极的较高迁移率中很明显。
