G6Pase

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Introduction

Both gluconeogenesis and glycogenolysis result in the formation of glucose 6-phosphate (Glc-6-P), which must be hydrolyzed by glucose 6-phosphatase (G6Pase) to form glucose to be released into the circulation. Therefore, G6Pase plays a key role in blood glucose homeostasis. Coris' work showed that glycogen is degraded to glucose 1-phosphate by phosphorolysis, which means that the phosphate must be removed at a later stage to generate free glucose. de Duve and co-workers showed that the liver contains a phosphatase that acts specifically on Glc-6-P. Subsequent cell grading studies have shown that G6Pase is associated with the endoplasmic reticulum. A further advance was the discovery that G6Pase deficiency causes glycogen storage disease type I (GSD I), and the hypothesis put forward by Arion and co-workers in 1975 that the catalytic site of G6Pase faces the ER lumen and that it requires transporters of 6-P, glucose and P_i.

Kinetic properties

The properties of G6Pase depend on the integrity of the microsomal membrane. In intact microsomes, it specifically catalyzes the hydrolysis of Glc-6-P, and its activity on this phosphate ester is more than 10-fold higher than that on mannose 6-phosphate (Man-6-P). Treatment with a variety of detergents, including anionic, cationic, and neutral detergents, modestly stimulates the hydrolysis of Glc-6-P, but significantly (10-15-fold) increased phosphatase activity on other substrates, such as Man-6-P, glucosamine 6-phosphate and 2-deoxyglucose 6-phosphate. G6Pase also hydrolyzes substrates other than sugar phosphates, such as PP_i and carbamoyl phosphates. As its reaction mechanism involves phosphatase intermediates, G6Pase also exhibits phosphotransferase activity capable of synthesizing Glc-6-P and carbamoyl phosphate from glucose and various phosphate donors such as PP_i and Man-6-P. These activities of G6Pase were more apparent in microsomes treated with detergent. It has been proposed that G6Pase can also participate in the phosphorylation of glucose by using carbamoyl phosphate as a phosphate donor when sugar concentrations are elevated (as in diabetes). However, G6Pase deficiency leads to severe hypoglycemia and its overexpression leads to glucose intolerance, suggesting that the physiological role of this enzyme is in glucose production. This conclusion is consistent with studies on isolated hepatocytes showing that the rate of glucose phosphorylation correlates with the activity of glucokinase.

Figure 1. Reaction mechanism of G6Pase (SCHAFTINGEN, E.V. 2002)

cDNA and gene

The cloning of the cDNA encoding G6Pase took advantage of the low-level expression of this enzyme in mice homozygous for radiation-induced albino mutation, which causes severe hypoglycemia in the first few hours after birth.  The activity of other hepatic enzymes, including phosphoenolpyruvate carboxykinase and glutamine synthase, was also reduced in these mice. Differential screening of the cDNA library resulted in the isolation of several clones that expressed weakly in the liver of homozygous lethal albino mutant mice. Expression of the cDNA in COS cells indicated that it indeed encodes G6Pase. Human cDNA encodes a protein of the same size as mouse G6Pase. Both are 10-12 kb long and contain five exons. The human gene is located at 17q21. The cDNA sequences from other species are also described, including rat, dog, and two species of fish.

Screening of a mouse insulinoma library yielded a cDNA encoding a protein 49% identical to liver G6Pase. This protein is also very hydrophobic and has a retention signal of the endoplasmic reticulum. Northern blots indicated that it is only expressed in the endocrine pancreas. The mouse, human and rat genes corresponding to this cDNA have been identified and their structures determined. Both mouse and human genes are located on chromosome 2 and have a five-exon structure like the G6Pase gene. The rat gene appears to be a pseudogene because exon 4 is missing and exons 1 and 5 are interrupted by frameshift mutations. The role, if any, of the product encoded by this gene in humans and mice is currently unclear.

Figure 2. Substrate-transport model (Arion’s model) (SCHAFTINGEN, E.V. 2002)