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 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.
The overall scheme of this protocol is outlined in Figure 1.
1. Solution Preparation for Standard Curves and Assays
2. Preparation of an Acetyl Phosphate Standard Curve
3. Assay for Acetate Kinase Activity
4. Representative Results:
The purpose of this assay is to measure acetate kinase activity in the direction of acetate formation. This is done by measuring consumption of the acetyl phosphate substrate, as there is no easy, direct measurement for acetate production. Acetyl phosphate remaining after a given time during the enzymatic reaction is converted to acetyl hydroxamate and subsequently to ferric hydroxamate complex, which has a reddish color (Figure 2) that can be measured at 540 nm. The standard curve plotting absorbance versus μmoles acetyl phosphate present in the standards is used to determine the amount of acetyl phosphate remaining in each reaction. The standard curve should be linear through the zero point. A representative standard curve shown in Figure 3 has a slope of 1.2332 absorbance units per μmole acetyl phosphate present and an R2 value of 0.99, indicating the data fits the linear equation well. The equation for the linear fit to the standard curve data is used for calculating the quantity of acetyl phosphate remaining in the reaction. The μmoles of acetyl phosphate consumed is determined as the difference between the μmoles of acetyl phosphate present in the control reaction lacking enzyme and the μmoles of acetyl phosphate remaining after the enzymatic reaction.
Purified, recombinant M. thermophila acetate kinase was assayed using the described protocol. For the experiment shown in Figure 4, the concentration of ADP was held constant at 5 mmol/L and the concentration of acetyl phosphate in the reaction was varied to determine the Km for acetyl phosphate. As expected, this experiment demonstrates that the enzyme follows standard Michaelis-Menten-like kinetics for each substrate and produced a hyperbolic saturation curve when plotting activity versus acetyl phosphate concentration. The apparent Km value for acetyl phosphate was 0.27 ± 0.01 mmol/L, which compares favorably to the published Km value of 0.47 ± 0.01 mmol/L using the coupled assay13.
The direct assay can also be used to measure activity of acetate kinases that utilize substrates other than ATP, which is not possible using the standard coupled assay. Purified recombinant E. histolytica acetate kinase16, which produces PPi rather than ATP in the acetate-forming direction, was subjected to this assay using sodium phosphate in place of ADP. The concentration of sodium phosphate in the reaction was held constant at 0.2 mol/L and the acetyl phosphate concentration was varied. As for the M. thermophila acetate kinase, the E. histolytica acetate kinase followed Michaelis-Menten-like kinetics for acetyl phosphate and a hyperbolic saturation curve was observed (Figure 5). The apparent Km value for acetyl phosphate was 0.50 ± 0.006 mmol/L. Reeves and Guthrie15 determined a Km value of 0.06 mmol/L for acetyl phosphate for the native enzyme; however, their enzyme was only partially purified and their assay involved four coupling enzymes which were also only partially purified. Thus, it is difficult to directly compare these values.
Protocol Scheme
Figure 1. Protocol Scheme
Figure 2. Acetyl phosphate standards. Increasing concentrations of acetyl phosphate were reacted with hydroxylamine and color was developed as described. The control lacking acetyl phosphate is bright yellow, and reactions containing increasing amounts of acetyl phosphate are successively darker in color. This color change is measured at 540 nm.
Figure 3. Sample acetyl phosphate standard curve. Varying amounts of acetyl phosphate were reacted with hydroxylamine and the color development solution and absorbance was measured at 540 nm. The absorbance at 540 nm versus μmoles acetyl phosphate was plotted and a linear fit to the data was applied.
Figure 4. Utilization of acetyl phosphate by the ATP-producing M. thermophila acetate kinase. Enzymatic activity was determined in the direction of acetate formation in the presence of 5 mmol/L ADP with varying concentrations of acetyl phosphate. The amount of acetyl phosphate consumed was determined as the difference between the starting amount of acetyl phosphate in the reaction and the amount of acetyl phosphate remaining after the enzymatic reaction. Assays were performed in triplicate with error bars shown.
Figure 5. Utilization of acetyl phosphate by the PPi-producing E. histolytica acetate kinase. Enzymatic activity in the direction of acetate formation was determined in the presence of 0.2 mol/L sodium phosphate with varying concentrations of acetyl phosphate. The amount of acetyl phosphate consumed was determined as the difference between the starting amount of acetyl phosphate in the reaction and the amount of acetyl phosphate remaining after the enzymatic reaction. Assays were performed in triplicate with error bars shown.
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 acetyl hydroxamate. The timing of the color development step and measurement of absorbance is less critical, and can be performed in as little as 5 minutes and up to 15 minutes following the addition of the hydroxylamine solution. The samples should be centrifuged before reading the absorbance, as the acidity of the sample may lead to precipitation of the enzyme and can produce a falsely high reading due to turbidity rather than actual color change. Breakdown of the developed reactions will begin to occur after 10-15 minutes.
The primary complications of this assay for examining enzyme kinetics are determination of the amount of enzyme to use and the length of time to run the first step of the reaction. These must be determined empirically for each enzyme such that the level of activity is not so high that all of the acetyl phosphate substrate is consumed. Careful attention should be given to the percent acetyl phosphate consumption for each reaction point in a kinetic curve. Generally, lower concentrations of the varied component on a kinetic curve should result in acetyl phosphate consumption up to 90 percent or slightly above, while consumption of acetyl phosphate at saturating substrate concentrations should approach 10 percent or slightly less. Once the enzyme concentration is optimized, the substrate range for determination of kinetic constants can then be determined.
Previous methods for measuring acetate kinase activity in the acetate-forming direction involved the use of coupled assays. These methods are problematic with respect to kinetic calculations due to dependence on the activity of the coupling enzyme(s), whose assay conditions (pH, ionic strength, temperature, etc.) may be incompatible with the optimal assay conditions for the enzyme of interest. In addition, use of a direct assay for studies with inhibitors may avoid issues that could arise from inhibition of the coupling enzymes. Overall, this assay should be useful not only for acetate kinase, but for any enzyme that utilizes acetyl phosphate or other activated short chain acyl substrates such as acetyl- or propionyl-CoA, propionyl phosphate, and acetyl-AMP, all of which are reactive with hydroxylamine. In these cases, the standard curve would be generated with the appropriate acyl substrate instead of with acetyl phosphate.
The authors have nothing to disclose.
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.
Name of the reagent | Company | Catalogue number | Comments |
Acetyl phosphate | Sigma Aldrich | 01409 | Lithium Salt (97% ) |
Sodium phosphate monobasic (dehydrate) | ThermoFisher | S381 | |
Sodium phosphate dibasic (anhydrous) | ThermoFisher | S374 | |
Magnesium chloride (hexahydrate) | ThermoFisher | M33 | |
Tris Base | ThermoFisher | B152 | |
Ferric chloride (hexahydrate) | ThermoFisher | I88 | |
Trichloroacetic acid | ThermoFisher | A324 | |
Hydroxylamine hydrochloride | ThermoFisher | H330 | |
Adenosine 5’-diphosphate sodium salt |
Sigma Aldrich | A2754 | |
Biomate III Spectrophotometer | ThermoFisher | 142982082 | Standard UV/Vis spectrophotometer |