Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates3

Different enzyme assays for ACTase study in H. pylori

ACTase properties were studied in situ in cell-free extracts to obtain information on enzyme function in an environment that was closer to the bacterium's physiological conditions. There are numerous and unique advantages in studying enzyme activities in purified preparations, but the use of simpler milieux may mask properties such as protein-protein interactions, that could be observed in more complex conditions. Investigation of an enzyme activity in crude extracts allows for its characterisation, and at the same time retains the possibility of discovering some of its interactions with other cell components.

Three different methods of studying ACTase activity were employed in this investigation. The results obtained by each technique were compared with the data from the others, to validate them and/or gather additional information. Comparison of the rates in Table 1, indicated that each method represented a valid procedure for studying ACTase activity in H. pylori.

NMR spectroscopy was the first method employed to identify ACTase in H. pylori. ¹H-NMR spectra showing the time-course of substrates and products of the ACTase reaction in incubations with H. pylori cell-free extracts were given in Figure 1 of the original article (1). Decrease of the peaks of the aspartate substrate, and appearance of peaks corresponding to a metabolic product were observed. The protons of the substrate carbamoyl phosphate are in chemical exchange with those of the aqueous solvent at rates which render them invisible in the ¹H-NMR spectrum under the experimental conditions employed. The ¹H-NMR resonances arising from H. pylori lysates or cell-free extracts were very small compared to those of the aspartate and the product, thus it was possible to follow the evolution of these metabolites over time. Assignment of the resonances from the product carbamoyl aspartate (CAA) was achieved by adding this metabolite to assay mixtures and comparing the spectral position of its resonances with those of the product in the bacterial preparations.

Metabolites have NMR spectral signatures characterized by their chemical structures. In the case of CAA the resonances arising from α-CH and β-CH2 groups of the aspartate moiety have chemical shifts and coupling constants different from those of the amino acid aspartate. Thus, it was possible to make an unambiguous assignment of CAA. This definite identification of a reaction product, is a feature lacking in other enzyme assays, particularly when complex preparations are assayed. For example, the radioactive or colorimetric assay measures the formation of a radioactive product and a ureido product, respectively, without yielding sufficient information about its identity. Although proper controls could ensure the validity of these methods, NMR spectroscopy gave direct evidence for the presence of ACTase activity. However, this technique has limitations, some of which are: a) sensitivity, because often relatively high concentrations of substrates are needed to obtain clear NMR signals; b) spectral overlap, under certain conditions some peaks may appear superimposed on others thus masking specific effects; and c) cost of operating NMR spectrometers, which may preclude their use in large scale enzyme analysis. Notwithstanding its limitations, this technique has proven most effective in the study of enzyme characteristics in H. pylori (4, 16-18).

Radioactive tracer analysis was another useful method to study ACTase activity in cell-free extracts. A main advantage of this technique is its sensitivity; measurement of radioactive decays allowed for determination of ACTase activity at relatively small enzyme concentrations. This could prove especially useful in studies on purified proteins, where yield is often quite low. Radioactive tracer analyses provided information on ACTase activity that could not be obtained by NMR analysis or the colorimetric assay. Specifically, to study the effects of carbamoyl aspartate, the ACTase end-product, on enzyme activity. In these experiments the added exogenous CAA will mask the product formed through the activity of the enzyme in both the NMR and colorimetric assays; the higher the concentration of added CAA, the less accurate are the measurements of the activity of the enzyme by NMR spectroscopy or spectrophotometry. The effects of CAA on ACTase activity were assessed confidently employing radioactive tracer analysis and, as shown in Figure 1, the observed product-inhibition of the enzyme activity was consistent with the inhibitory effects CAA had on H. pylori growth.

Because the labelled carbamoyl phosphate is used only in trace amounts and is an unstable compound it is very important to monitor its purity continuously. At one point in the work, significant radioactive counts were measured in samples only containing [¹⁴C]carbamoyl phosphate, even after addition of strong acid and heating. As mentioned in the methods, this treatment should allow for the removal of any unreacted [¹⁴C]carbamoyl phosphate as ¹⁴CO₂. The finding indicated the presence of an acid-stable contaminant in the stock [¹⁴C]carbamoyl phosphate. Apart from the obvious expense in obtaining fresh radioactive substrate, the possibility of further contamination and false positive results suggested this method may not the most appropriate for large scale characterisation of ACTase activity. Another less technical disadvantage of this technique is the safety in the handling of radioactive products. Although due care should prevent any problems, the element of risk involved may favour the use of a technique with less hazardous materials.