This work was supported by NSF award # 0920274 and South Carolina Experiment Station Project (SC-1700340) to KSS. This paper is Technical Contribution No. 5929 of the Clemson University Experiment Station.

The overall scheme of this protocol is outlined in Figure 1.
1. Solution Preparation for Standard Curves and Assays
<ol>
	<li>Prepare 100 mL of a 2 mol/L solution of hydroxylamine-HCl. Weigh out 13.9 g of hydroxylamine hydrochloride (MW 69.49 g/mol) and dissolve in approximately 50 mL distilled-deionized water (ddH2O). Adjust the pH to 7.0 using potassium hydroxide pellets or a concentrated solution. Bring the final volume to 100 mL. The solution can be stored at room temperature ...

<ol>
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A. 89, 7290-7294 (1992).</li><li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+of+similar+enzymatic+function+on+different+protein+folds:+the+hexokinase,+ribokinase,+and+galactokinase+families+of+sugar+kinases.">Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.</a> Protein Sci. 2, 31-40 (1993).</li><li>Buss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urkinase:+structure+of+acetate+kinase,+a+member+of+the+ASKHA+superfamily+of+phosphotransferases.">Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases.</a> J. Bacteriol. 183, 680-686 (2001).</li><li>Hurley, J. 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Chem. 159, 21-28 (1945).</li><li>Lipmann, F., Jones, M. E., Black, S., Flynn, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+ATP-CoA-acetate+reaction.">The mechanism of the ATP-CoA-acetate reaction.</a> J. Cell. Physiol. Suppl. 41, 109-112 (1953).</li><li>Rose, I. A., Grunberg-Manago, M., Korey, S. F., Ochoa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+phosphorylation+of+acetate.">Enzymatic phosphorylation of acetate.</a> J. Biol. Chem. 211, 737-756 (1954).</li><li>Allen, S. H., Kellermeyer, R. W., Stjernholm, R. L., Wood, H. 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The detection of acetyl phosphate in this assay is dependent upon a sufficient concentration of hydroxylamine hydrochloride and the concentration and acidity of the ferric chloride solution. Alteration of the assay volume will require reconsideration of both of these components. The enzymatic reactions described here were performed at 37&deg;C with the hydroxylamine termination performed at 60 &deg;C for 5 minutes. This higher temperature is critical to allow for rapid conversion of the remaining acetyl phosphate to acet...

<table border="1">
 <tr>
 <td width="160">Name of the reagent</td>
 <td width="108">Company</td>
 <td width="134">Catalogue number</td>
 <td width="103">Comments</td>
 </tr>
 <tr>
 <td>Acetyl phosphate</td>
 <td>Sigma Aldrich</td>
 <td>01409</td>
 <td>Lithium Salt (97% )</td>
 </tr>
 <tr>
 <td>Sodium phosphate monobasic (dehydrate)</td>
 <td>ThermoFisher</td>
 <td>S381</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium phosphate dibasic (anhydrous)</td>
 <td>ThermoFisher</td>
 <td>S374</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnesium chloride (hexahydrate)</td>
 <td>ThermoFisher</td>
 <td>M33</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>ThermoFisher</td>
 <td>B152</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ferric chloride (hexahydrate)</td>
 <td>ThermoFisher</td>
 <td>I88</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trichloroacetic acid</td>
 <td>ThermoFisher</td>
 <td>A324</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hydroxylamine hydrochloride</td>
 <td>ThermoFisher</td>
 <td>H330</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Adenosine 5&rsquo;-diphosphate 
 sodium salt</td>
 <td>Sigma Aldrich</td>
 <td>A2754</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biomate III Spectrophotometer</td>
 <td>ThermoFisher</td>
 <td>142982082</td>
 <td>Standard UV/Vis spectrophotometer</td>
 </tr>
</table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

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