Mattice Amanda M S, MacLean Isabelle A, Childers Christine L, Storey Kenneth B
Institute of Biochemistry, Department of Biology, Carleton University, Ottawa, Canada.
Institute of Biochemistry, Department of Biology and Chemistry, Carleton University, Ottawa, Canada.
PeerJ. 2018 Jun 8;6:e4918. doi: 10.7717/peerj.4918. eCollection 2018.
Pyruvate kinase (PK) is responsible for the final reaction in glycolysis. As PK is a glycolytic control point, the analysis of PK posttranslational modifications (PTM) and kinetic changes reveals a key piece of the reorganization of energy metabolism in an anoxia tolerant vertebrate.
To explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia.
Both muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while L-alanine was only reduced in the liver. Both isozymes showed significant increases in threonine phosphorylation (by 42% in muscle and 60% in liver) and lysine methylation (by 43% in muscle and 70% in liver) during anoxia which have been linked to suppression of PK activity in other organisms. Liver PK also showed a 26% decrease in tyrosine phosphorylation under anoxia.
Anoxia responsive changes in turtle muscle and liver PK coordinate with an overall reduced activity state. This reduced affinity for the forward glycolytic reaction is likely a key component of the overall metabolic rate depression that supports long term survival in anoxia tolerant turtles. The coinciding methyl- and phospho- PTM alterations present the mechanism for tissue specific enzyme modification during anoxia.
丙酮酸激酶（PK）负责糖酵解的最后一步反应。由于PK是糖酵解的一个控制点，对PK翻译后修饰（PTM）和动力学变化的分析揭示了耐缺氧脊椎动物能量代谢重组的关键环节。
为了探究PK的调控机制，从有氧和暴露于缺氧环境20小时的红耳龟的红色骨骼肌和肝脏中分离出该酶。采用动力学分析和免疫印迹法评估缺氧期间酶的功能以及酶结构的相应共价修饰。
缺氧期间，肌肉和肝脏同工型对磷酸烯醇丙酮酸底物的亲和力均降低，且肌肉PK对ADP的亲和力也较低。缺氧暴露后，两种组织中PK对抑制剂ATP和乳酸的Ki值均降低而L-丙氨酸仅在肝脏中降低。缺氧期间，两种同工酶的苏氨酸磷酸化（肌肉中增加42%，肝脏中增加60%）和赖氨酸甲基化（肌肉中增加43%，肝脏中增加70%）均显著增加，这与其他生物体中PK活性的抑制有关。缺氧时肝脏PK的酪氨酸磷酸化也降低了26%。
乌龟肌肉和肝脏PK的缺氧反应性变化与整体活性状态降低相协调。对正向糖酵解反应亲和力的降低可能是支持耐缺氧乌龟长期生存的整体代谢率降低的关键组成部分。同时出现的甲基化和磷酸化PTM改变揭示了缺氧期间组织特异性酶修饰的机制。
