2.2.3. Substrate Specificity of MTase

2.2.2. MTase Activity in the Presence of Guanosine Triphosphate (GTP) as a Substrate The MTase kinetics were performed in the presence of GTP as its substrate while keeping the concentrations of S-adenosyl methionine (SAM) (methyl donor) and the enzyme constant at 1 μM and 0.675 μM, respectively. The concentration of the GTP substrate (methyl acceptor) ranged from 0 to 10 mM. The Michaelis–Menten equation was used to calculate the km value for GTP, which was found to be 0.387 mM (Figure 3A). The km value was also determined using a Lineweaver–Burke plot produced by GraphPad Prism 9. The straight-line equation for the plot, Y = 0.0003207 × X + 0.0008274, was used to calculate the km and Vmax values (Figure 3B). The calculated km and Vmax values from the Lineweaver– Burke plot were found to be 0.387 mM and 120,496, respectively. The substrate specificity of the enzyme was studied using different nucleotides, cytidine triphosphate (CTP), uridine triphosphate (UTP), cyclic adenosine monophosphate (cAMP), guanosine diphosphate (GDP), GTP, and deoxy guanosine triphosphate (dGTP), as well as the capped analogues, 7-methyl guanosine diphosphate (m<sup>7</sup> -GDP) and 7-methyl-guanosine triphosphate (m<sup>7</sup> -GTP). The reaction was performed, in which 0.675 µM of MTase was tested for enzyme activity in the presence of 0.5 mM of different nucleotide triphosphates (NTPs) as cap analogues, as mentioned above. The concentration of SAM, the methyl donor, was kept constant at 1 µM in the reactions. The activity assay demonstrated that GDP is a better substrate than GTP, while minimal activity was observed for CTP, UTP, cAMP, and dGTP (Figure 4). The cap analogues, m<sup>7</sup> -GDP and m<sup>7</sup> -GTP, showed minimal activity. These results showed the specificity of MTase activity, indicating that there must be a free position on the substrate to transfer the methyl group. The minimal activity for m<sup>7</sup> -GDP and m<sup>7</sup> -GTP was observed since the N-7 position of the guanosine is already methylated [8] (Figure 4).

2.2.3. Substrate Specificity of MTase

**Figure 3. Kinetic studies of MTase:** (**A**). The reaction mixture contained 1 μM SAM and different concentrations of GTP. Each data point represents the mean value, and the error bars indicate the standard deviation. The Km value of the substrate, GTP, was calculated using the Michaelis–Menten equation and found to be approximately 0.38 mM (**B**). The Lineweaver–Burke plot was produced by GraphPad Prism 9. A straight-line equation was determined using Km and Vmax (Y = 0.0003207 × X + 0.0008274). The calculated Km and Vmax values using the Lineweaver–Burke plot were 0.387 mM and 120,496, respectively. **Figure 3. Kinetic studies of MTase:** (**A**) The reaction mixture contained 1 µM SAM and different concentrations of GTP. Each data point represents the mean value, and the error bars indicate the standard deviation. The K<sup>m</sup> value of the substrate, GTP, was calculated using the Michaelis–Menten equation and found to be approximately 0.38 mM. (**B**) The Lineweaver–Burke plot was produced by GraphPad Prism 9. A straight-line equation was determined using K<sup>m</sup> and Vmax (Y = 0.0003207 × X + 0.0008274). The calculated K<sup>m</sup> and Vmax values using the Lineweaver–Burke plot were 0.387 mM and 120,496, respectively. *Molecules* **2022**, *27*, x FOR PEER REVIEW 6 of 18

**Figure 4. Effect of different substrates on HEV MTase activity:** The graph represents the activity of MTase using different substrate analogues. The bars represent various nucleotide analogues, as indicated in the figure. The values have been normalised to the percentage activity of the enzyme in the presence of GTP. The enzyme assay was performed in triplicate; the graph represents the mean value while the error bar indicates the standard deviation. The statistical significance of the data was determined using Student's *t*-test. \*\* *p*-value < 0.05, \*\*\* *p*-value < 0.0005. **Figure 4. Effect of different substrates on HEV MTase activity:** The graph represents the activity of MTase using different substrate analogues. The bars represent various nucleotide analogues, as indicated in the figure. The values have been normalised to the percentage activity of the enzyme in the presence of GTP. The enzyme assay was performed in triplicate; the graph represents the mean value while the error bar indicates the standard deviation. The statistical significance of the data was determined using Student's *t*-test. \*\* *p*-value < 0.05, \*\*\* *p*-value < 0.0005.

Although divalent cations are crucial for the activity of many viral enzymes, no study has been observed the role of divalent cations on MTase activity. Hence, MTase activity in response to varying concentrations of Mg2+ was studied. This resulted in the linear in-

the enzyme, but no effect on MTase activity was seen (data not shown). To further validate the impact of Mg2+, the reaction was performed in the presence of EDTA, which is supposed to be a chelating agent for the magnesium. When increasing the concentration of EDTA from 0 to 8 mM, the enzyme activity decreased significantly, establishing that the

presence of magnesium is essential for the MTase activity (Figure 5B).

2.2.4. Effect of Magnesium (Mg2+) on MTase Activity
