FBPase

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Introduction

Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis, and a potential drug target for the treatment of type II diabetes. The protein is also related to some cancer cells lacking FBPase activity which promotes glycolysis facilitating the Warburg effect. Therefore, due to its important role in diabetes and related cancers, more and more researches on this enzyme have been conducted. The mammalian enzyme is tetrameric, which is competitively inhibited by fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the enzyme's active site. Several studies have shown that two residues (Lys112 and Tyr113) in the AMP binding site are involved in initiating the message between the two sites. This tyrosine residue has recently been shown to be important for protein interactions with the antidiabetic drug metformin. In short, these findings may have a profound impact on the design of new FBPase inhibitors or activators.

Fructose 1,6-bisphosphatase and disease

Fructose 1,6-bisphosphatase (fructose diphosphatase; FBPase; EC 3.3.1.11) is responsible for catalyzing the hydrolysis of fructose 1,6-diphosphate to fructose 6-phosphate in gluconeogenesis and the Calvin cycle. Although ATP is required to phosphorylate fructose 6-phosphate during glycolysis, ATP is not produced during dephosphorylation during gluconeogenesis. This enzyme is also a key regulatory point and is inhibited by fructose 2,6-diphosphate and AMP. Under the condition of low cellular ATP concentration, FBPase is relatively inactive and ATP synthesis is stimulated, which avoids the "futile cycle".

Figure 1. Conversion between fructose 1,6-diphosphate and fructose 6-phosphate

FBPase is a homotetramer in most species, with yeast being a notable exception. Like many oligomeric enzymes, this enzyme exhibits allosteric behavior. The binding of fructose 2,6-bisphosphate to the active site sterically hinders the access of the substrate fructose 1,6-bisphosphate. AMP binds at a separate site far away from the active site, and its binding promotes the conformational change of the enzyme. The two subunits rotate about 19° relative to the other two subunits, resulting in reduced enzyme activity.

The role of FBPase in diabetes has led to it being suggested as a potential treatment for type 2 diabetes. In this disease, gluconeogenesis is an important factor leading to excess glucose, and reducing this excess glucose will alleviate the pathology associated with high glucose concentrations in blood and tissues. And FBPase has an attractive advantage that its inhibition will only affect gluconeogenesis and not glycolysis. In addition, the presence of natural allosteric regulation of the enzyme suggests that it may mimic the effects of AMP, thereby significantly reducing its activity. Therefore, drug discovery efforts have focused on identifying molecules that recognize AMP binding sites, induce allosteric inhibition of FBPase, but do not interact with other adenosine nucleotide binding enzymes. FBPase is associated with a rare autosomal recessive inherited metabolic disease (OMIM #229700). The patient's gluconeogenesis is impaired, so symptoms such as hypoglycemia, ketosis and lactic acidosis occur. If the early diagnosis is a timely and aggressive treatment, a good prognosis can be obtained. These measures include avoiding fasting, enrichment of the diet in glucose, and reducing the fructose in the diet. But if left untreated, it can be fatal in newborn babies.

Getting the message across

The existence of allostery in FBPase infers that there is an information transmission pathway within the protein. Generally, allosteric changes are transmitted through proteins by conformational changes, alterations in mobility, or a combination of the two. The crystal structure of FBPase with or not with AMP bound provides a wealth of information about the changes in the interface between the ligand-binding site and the tetramer subunit. One of the results of these structural and dynamic changes is the displacement of a loop in the active site, which is essential for enzyme catalysis, and its distance from the substrate-binding site will reduce the enzyme activity.

The study of Topaz et al. focused on seven variants. As expected, changes in the two lysine residues in the AMP binding site resulted in decreased reactivity to the compound’s modulation, but did not affect regulation by fructose 2,6-bisphosphate. These variants also have the effect of increased catalytic efficiency (measured by the specificity constant kcat / Km). It has recently been reported that the Y113F variant (Y114F in the numbering scheme used in the paper reporting this variant) is greatly reduced in sensitivity to AMP, but the sensitivity to fructose 2,6-bisphosphate is also greatly reduced, indicating that this is the key region in AMP binding site. It seems that these residues are not only involved in the binding of AMP, but also in the initiation of the transmission of information to the active site.