­Characterization of pyruvate kinase from the anoxia tolerant turtle, Trachemys scripta elegans: a potential role for enzyme methylation during metabolic rate depression

Abstract

BackgroundPyruvate 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.MethodsTo explore PK regulation, the enzyme was isolated from red skeletal muscle and liver of aerobic and 20-hr anoxia-exposed red eared-slider turtles (Trachemys scripta elegans). Kinetic analysis and immunoblotting were used to assess enzyme function and the corresponding covalent modifications to the enzymes structure during anoxia.ResultsBoth muscle and liver isoforms showed decreased affinity for phosphoenolpyruvate substrate during anoxia, and muscle PK also had a lower affinity for ADP. I50 values for the inhibitors ATP and lactate were lower for PK from both tissues after anoxic exposure while I50 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.DiscussionAnoxia 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.

New Insights to Regulation of Fructose-1,6-bisphosphatase during Anoxia in Red-Eared Slider, Trachemys scripta elegans

“…Being reversible processes, PTMs provide an economical solution to alter enzyme activity as opposed to more radical energy-expensive solutions, such as the proteolysis/resynthesis of enzymes over the anoxia/reoxygenation cycle. Changes in the enzyme activities and catalytic properties due to PTMs in response to environmental stresses have been observed in multiple previous studies conducted on both vertebrate and invertebrate species [23,45,[56][57][58]. Considering the above results, and previous studies, it can be said that PTMs are an important factor behind the differential activity of an enzyme under aerobic versus anoxic conditions.…”

“…Threonine phosphorylation has been reported to suppress enzyme activities during anoxia and other forms of hypometabolism. For example, previous studies on the marine mollusk, Busycon canaliculatum [44], and the red-eared slider [45] showed a correlation between increased pyruvate kinase phosphorylation and changes in enzyme properties that would suppress enzyme function. Furthermore, based on an electrophoretic analysis of [ 32 P] incorporation, it was suggested that O-phosphothreonine residue(s) were produced under anoxia [46].…”

MicroRNAs regulate survival in oxygen-deprived environments

“…Under such conditions, these processes have been found to be differentially regulated in a tissue-specific manner in response to the unique metabolic requirements of each organ (Ziello et al, 2007). For instance, anoxia-induced responses have been shown to be tissue specific in various other models of hypoxia and anoxia tolerance, such as turtles, frogs and squid (Mattice et al, 2018;Al-attar et al, 2017;Gerber et al, 2016;Hadj-Moussa et al, 2018;Krivoruchko and Storey, 2015).…”

MicroRNA regulation and expression in the anoxia-tolerant red-eared slider turtle, Trachemys scripta elegans

“…Several studies on kinases in the liver of red-eared slider turtles have shown decreased kinase activity after prolonged anoxia exposure, supporting the present sequencing results indicating kinase downregulation (Mattice et al, 2018;Mehrani & Storey, 1995, 1996.…”