Salvucci M E
United States Department of Agriculture-Agricultural Research Service, University of Kentucky, Lexington 40546-0076, USA.
Plant Physiol. 1993 Oct;103(2):501-8. doi: 10.1104/pp.103.2.501.
Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.
利用水溶性N-羟基琥珀酰亚胺酯对核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）活化酶进行化学修饰，以鉴定对活性至关重要的反应性赖氨酸残基。将Rubisco活化酶与磺基琥珀酰亚胺基-7-氨基-4-甲基香豆素-3-乙酸酯（AMCA-磺基-NHS）或磺基琥珀酰亚胺基乙酸酯（磺基-NHS-乙酸酯）一起孵育，会导致ATP酶活性逐渐失活，并同时丧失激活Rubisco的能力。AMCA-磺基-NHS是Rubisco活化酶更有效的失活剂，其失活的二级速率常数为239 M-1 s-1，而磺基-NHS-乙酸酯为21 M-1 s-1。AMCA-磺基-NHS使酶活性失活与每摩尔42-kD Rubisco活化酶单体掺入1.9摩尔AMCA相关。ADP是Rubisco活化酶的竞争性抑制剂，可提供相当大的保护以防止Rubisco活化酶失活，并减少掺入Rubisco活化酶单体中的AMCA量。对来自AMCA-磺基-NHS修饰酶的主要标记肽进行序列分析表明，修饰的主要位点是四肽甲硫氨酸-谷氨酸-赖氨酸-苯丙氨酸中的赖氨酸-247（K247）。当ATP酶活性完全失活时，K247的修饰占每摩尔Rubisco活化酶单体掺入1摩尔AMCA。用在腺嘌呤碱基或γ-磷酸上衍生化的ATP类似物对AMCA-磺基-NHS和磺基-NHS-乙酸酯修饰的Rubisco活化酶进行光亲和标记表明，K247本身对于腺嘌呤核苷酸的结合不是必需的。相反，数据表明K247的必要性可能是由于这个高反应性、物种不变的残基参与了催化过程中蛋白质与核苷酸磷酸之间发生的强制性相互作用。
