Regulation of tail muscle hexokinase in the anoxia-tolerant freshwater crayfish, Orconectes virilis

Hexokinase (HK)(E.C.2.7.1.1) is the enzyme responsible for catalyzing the first step of glucose metabolism, the phosphorylation of glucose to glucose-6-phosphate (G6P). The present study investigated HK from tail muscle of the freshwater crayfish Oconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20 h anoxic). Evidence indicated that HK was converted to a low phosphate form under anoxia. ProQ Diamond phosphoprotein staining showed a 39% higher bound phosphate content on aerobic HK compared with the anoxic form, yet treatment of aerobic HK with treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1+PP2A, PP2B, and PP2C) yielded no significant changes in kinetic parameters. By contrast, investigation of the stability and bound fractions of aerobic verses anoxic HK yielded stark differences in both susceptibility to urea denaturation and subsequent proteolytic cleavage, as well as a decrease in the amount of enzyme in the bound state. The physiological consequence of anoxiainduced HK dephosphorylation may be to stabilize and release HK during anoxia, increasing the glycolytic capacity of the animal. © 2012 Dawson et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence: [email protected] 1 Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada. Background Hexokinase (HK)(E.C.2.7.1.1) is the enzyme responsible for catalyzing the first step of glucose metabolism, the phosphorylation of glucose to glucose-6-phosphate (G6P): D-Glucose + ATP → D-Glucose-6-phosphate + ADP + H+ Glucose is a key source of energy for living organisms and is delivered by the blood to all organs of the body. Once transported into cells, glucose is rapidly phosphorylated by HK to form G6P and this allows the sugar to be directed into many different pathways such as; glycolysis to produce ATP, the pentose phosphate pathway to form NADPH and various sugar phosphates, or glycogen as a fuel storage [1]. There have been 4 isozymes reported in mammals, HK I-IV, which are found in different tissues and locations within the cell [2,3]. HK IV, otherwise known as glucokinase, is liver-specific and is primarily responsible for storing excess sugar into glycogen reserves; it has a high Km for glucose. HK I-III all share a similar molecular weight (~100 kDa) and much lower Km values for glucose (< 1 mM). HK I-III have been separated by ion exchange chromatography and isoelectric focusing [2,3]. HK isozymes I-III can bind to the outer membrane of mitochondria via an association with the porin that is located on the outer surface of the mitochondria [4]. The isozymes exhibit different kinetic parameters such as their substrate affinities for ATP and glucose, as well as their susceptibility to product inhibition by G6P [1]. Crayfish have a significant capacity for long term survival under anoxic conditions by switching to anaerobic glycolysis as their primary ATP-generating pathway and buffering lactate accumulation by Ca2+ release from their carapace [5]. Past studies on the regulation of glycolytic enzymes have demonstrated that, in cancer, a delicate interaction between the transcription factors MYC and HIF cause a differential expression of HK II [6]. As MYC levels decrease and HIF expression increase under low oxygen conditions, HK is upregulated. In addition to altered amounts of HK protein under low oxygen conditions, HK could also be regulated by posttranslational or allosteric mechanisms to alter its activity and/or function under high versus low oxygen conditions. Given the well-developed anaerobic capacity of crayfish, this model would be a good one in which to assess the effects of anoxia on HK regulation, particularly in light of the results from previous work showing anoxia-responsive regulation of other key enzymes including arginine kinase and glutamate dehydrogenase in crayfish muscle [7,8]. Previous studies in our lab have shown that one of the mechanisms of HK regulation in response to stress is reversible protein phosphorylation. HKI and II from the skeletal muscle of hibernating ground squirrels [9] and HK from the skeletal muscle of freeze-tolerant frogs [10] was shown to be regulated by reversible phosphorylation. Given the role of HK in gating glucose entry into glycolysis and the particular importance of HK to energy metabolism under anoxic conditions, as well as the evidence from previous studies of differential regulation of HK in response to stress, the current work investigates the regulation of HK during anoxia in the tail muscle of O. virilis.