Kanade Santosh R, Paul Beena, Rao A G Appu, Gowda Lalitha R
Department of Protein Chemistry and Technology, Central Food Technological Research Institute (CFTRI), Mysore-570 020, India.
Biochem J. 2006 May 1;395(3):551-62. doi: 10.1042/BJ20051509.
Field bean (Dolichos lablab) contains a single isoform of PPO (polyphenol oxidase)--a type III copper protein that catalyses the o-hydroxylation of monophenols and oxidation of o-diphenols using molecular oxygen--and is a homotetramer with a molecular mass of 120 kDa. The enzyme is activated manyfold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid-pH. The enhancement of kcat upon activation is accompanied by a marked shift in the pH optimum for the oxidation of t-butyl catechol from 4.5 to 6.0, an increased sensitivity to tropolone, altered susceptibility to proteolytic degradation and decreased thermostability. The Stokes radius of the native enzyme is found to increase from 49.1+/-2 to 75.9+/-0.6 A (1 A=0.1 nm). The activation by SDS and acid-pH results in a localized conformational change that is anchored around the catalytic site of PPO that alters the microenvironment of an essential glutamic residue. Chemical modification of field bean and sweet potato PPO with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide followed by kinetic analysis leads to the conclusion that both the enzymes possess a core carboxylate essential to activity. This enhanced catalytic efficiency of PPO, considered as an inducible defence oxidative enzyme, is vital to the physiological defence strategy adapted by plants to insect herbivory and pathogen attack.
豇豆（Dolichos lablab）含有一种多酚氧化酶（PPO）的单一同工型——一种III型铜蛋白，它利用分子氧催化单酚的邻羟基化和邻二酚的氧化——并且是一种分子量为120 kDa的同四聚体。该酶在低于其临界胶束浓度的阴离子洗涤剂SDS存在下或暴露于酸性pH时会被激活许多倍。激活后kcat的增强伴随着叔丁基儿茶酚氧化的最适pH从4.5显著移至6.0，对托酚酮的敏感性增加，对蛋白水解降解的敏感性改变以及热稳定性降低。发现天然酶的斯托克斯半径从49.1±2增加到75.9±0.6 Å（1 Å = 0.1 nm）。SDS和酸性pH的激活导致局部构象变化，该变化锚定在PPO的催化位点周围，改变了一个必需谷氨酸残基的微环境。用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺对豇豆和甘薯PPO进行化学修饰，然后进行动力学分析，得出结论：这两种酶都具有对活性至关重要的核心羧酸盐。PPO这种增强的催化效率，被认为是一种可诱导的防御氧化酶，对于植物适应昆虫食草和病原体攻击的生理防御策略至关重要。
