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

Summary

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.

Abstract

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, 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 thermophila^7-14. An acetate kinase which can only utilize PP_i 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 genus^15,16.

In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, 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 dehydrogenase^21,22, 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 PP_i.

Protocol

The overall scheme of this protocol is outlined in Figure 1.

1. Solution Preparation for Standard Curves and Assays

1. 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 for up to 30 days or at 4 °C for up to 90 days.
2. Prepare 100 mL of a ferric chloride/ hydrochloric acid solution. Weigh out 13.5 g ferric chl....

Discussion

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°C with the hydroxylamine termination performed at 60 °C for 5 minutes. This higher temperature is critical to allow for rapid conversion of the remaining acetyl phosphate to acet.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetyl phosphate	Sigma-Aldrich	01409	Lithium Salt (97% )
Sodium phosphate monobasic (dehydrate)	Thermo Fisher Scientific, Inc.	S381
Sodium phosphate dibasic (anhydrous)	Thermo Fisher Scientific, Inc.	S374
Magnesium chloride (hexahydrate)	Thermo Fisher Scientific, Inc.	M33
Tris Base	Thermo Fisher Scientific, Inc.	B152
Ferric chloride (hexahydrate)	Thermo Fisher Scientific, Inc.	I88
Trichloroacetic acid	Thermo Fisher Scientific, Inc.	A324
Hydroxylamine hydrochloride	Thermo Fisher Scientific, Inc.	H330
Adenosine 5’-diphosphate sodium salt	Sigma-Aldrich	A2754
Biomate III Spectrophotometer	Thermo Fisher Scientific, Inc.	142982082	Standard UV/Vis spectrophotometer