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From Proteopedia

Introduction

Asparagine Synthetase (ASNS) is an enzyme that catalyzes the conversion of Aspartic Acid to Asparagine through an ATP dependent amination reaction, using Mg2+ as a co-factor. ASNS can be found in both plants and mammals, however, in plants there are two forms ASNS-A and ASNS-B^[1]. Both forms use the same starting molecule of Aspartic Acid and produce Asparagine, the only difference coming from the source of the Nitrogen. ASNS-A uses inorganic Nitrogen in the form of ammonia or diatomic Nitrogen and ASNS-B uses Glutamine as its Nitrogen source; ASNS-B will be the focus of this page because it is very similar and undergoes the same reaction as ASNS in mammals. ASNS is in almost every somatic mammalian cell but is expressed in exponentially higher concentrations in the pancreas.

Structure

Asparagine Synthetase is a homodimer comprised of two domains ligated together. ASPS consists of a (residues 2-191) and and complex (residues 213-536). There are two specific domains that contain the substrate binding sites. The N-terminal domain which contains two layers of anti-parallel beta sheets comprised of six layers each, this is where the Glutamine binds. A C-terminal domain also exists, it is comprised of five parallel beta sheets with alpha helices on either side of it, ^[2] this is where the Mg2+, ATP and Aspartic Acid bind. The two active sites contained within the terminal domains are linked together by a tunnel of hydrophobic and polar surface amino acid residues. Specifically is important for the binding of the Beta-Asparty-AMP intermediate into the enzyme.^[3]

Amino Acid Composition

The Entire amino acid sequence for this enzyme complex can be found below. ASNS consists 1662 residues or 554 amino acids with a molecular weight of 62.7 kDa.

Function

Asparagine Synthetase catalyzes the interconversion of Aspartic Acid and Glutamine to Asparagine and Glutamic Acid. This is not a straight forward transferase reaction since the interconversion is not directly between Aspartic Acid and Glutamine. Instead this reaction more closely resembles a two part ATP dependent ligase reaction. ASNS has two distinct pockets; one where Adenosine Triphosphate (ATP) binds and is stabilized by the hydrogen bonds it forms with Ser 346 (Gamma Oxygen) and amide groups of Val 272, Leu 232 and Gly 347. This binding of ATP to the N-Terminal domain is to stabilize the intermediate it forms with the Aspartic Acid; Beta-Aspartyl AMP (BAspAMP). This intermediate complex has to bind to the ASNS enzyme before the Glutamine binds into its pocket to establish coordination of the binding sites within the ASNS enzyme. After the BAspAMP intermediate binds in its pocket and establishes the coordination of the enzyme the Glutamine binds to its pocket in the C-Terminal domain and is stabilized specifically by its bonds with Arg 49, Asn 74, Glu 76, and Asp 98 residues. Once the substrates have been bound and stabilized free water hydrolyzes the amine group on the side chain of Glutamine causing the newly formed ammonia (NH3) group to detach leaving a carboxylate ion as the new functional group converting the Glutamine to Glutamate. The free Ammonia molecule then binds with the carbonyl carbon of the BAspAMP complex forcing the carbonyl carbon of the Aspartic Acid residue to favor the amine group both electrochemically and sterically and break its bond with the AMP releasing it, inorganic phosphate and forming a free Asparagine molecule, excess water in the environment then protonates the Glutamate into a free form Glutamic Acid molecule. This can be seen in the above right figure. The main difference between ASNS-B and ASNS-A is that instead of using Glutamine for the Nitrogen source for this reaction ASNS-A uses ammonia or diatomic Nitrogen directly.

Future Research and Prospective Applications For Asparagine Synthetase

Leukemia Treatment

L-Asparaginase is a naturally occurring enzyme in bacteria that breaks down Asparagine. This easy breakdown of Asparagine into Aspartic Acid and Ammonia by L-Asparagine is able to suppress tumor cell growth cause by lymphoblastic leukemia cells by depleting the Asparagine stores creating a shortage in the plasma of the cell that cannot be compensated for potentially leading to apoptosis of the cell.^[4]Sometimes L-Asparaginase activity does not work to suppress the tumor cells. A recent study has been conducted to see if what could potentially cause some cells to be susceptible to treatment and others not be from within the same system. Leukemia infected cell lines were analyzed to see which ones were L-Asparaginase treatment immune and then those cell lines analyzed for protein expression and composition. It was found that in the cell lines that were immune to treatment there was an over expression of ASNS and therefore, an over expression of Asparagine to be used for maintenance and growth of the cells plasma.^[5] These findings are being evaluated and a potential new approach to treat lymphoblastic leukemia by using techniques such as RNAi silencing to suppress ASNS activity and allow L-Asparaginase to induce apoptosis in the cancerous cells.

Leukemia Tumor Suppression

A recent study has shown that a using a adenylated sulfoxmine transition-state analogue 1 has the ability to inhibit human ASNS. This inhibition of the continual production of Asparagine has also been able to suppress the growth of L-Asparaginase resistant Leukemia mouse cell lines.^[6]

Ovarian Cancer Predictive Bio-Marker

Recent studies have been trying to find a potential link between the amount of ASNS activity in certain cell cultures as a precursor for L-Asparaginase activity. L-Asparaginase is an enzyme that degrades Asparagine and has been used since the 1970's in basic cancer treatment plans for acute lymphoblastic leukemia.^[7]The body will naturally secrete L-Asparaginase as a response to a potential forming tumor or potential cancer cell aggregation. These studies are finding a correlation between the amount of ASNS being expressed prior to the expression of L-Asparaginase synthetase,^[8] since L-Asparaginase has only been shown to help combat lymphoblastic leukemia this would not be a direct treatment but rather an indicator that something is happening that requires attention and hopefully allow doctors to screen for ASNS activity and catch ovarian cancer in it's earlier stages when it is more treatable and easier to remove from the body.

References