*2.2. MTase Activity of 37 kDa Protein*

### *2.2. MTase Activity of 37 kDa Protein*  2.2.1. Enzyme Kinetics

2.2.1. Enzyme Kinetics MTase kinetics were studied as described in Materials and Methods to determine the nature of the enzyme and its activity. The activity of the purified protein was determined through different parameters, including various ranges of temperature, pH, and time. A linear increase in the enzyme activity was observed upon the increase in MTase concentration from 0–5 μM (Figure 2A). Further, enzyme activity was studied in the pH range from 6 to 12, leading to a bell-shaped curve with an optimal pH of 8.0 (Figure 2B). Additionally, the optimal temperature of the enzyme activity was found to be 37 °C (Figure 2C), above which the enzyme became denatured. Further, through the time-course assay, the optimal enzyme activity was found at 150 min, after which it plateaued (Figure 2D). For all future studies, the concentration of the enzyme used was 0.675 μM, while the concentration of GTP used was 0.4 mM. The graphs were plotted in terms of S-adenosyl homocysteine (SAH) concentration, determined using the standard curve. All the reactions were performed in triplicate, and the readings were subtracted from those of the no en-MTase kinetics were studied as described in Materials and Methods to determine the nature of the enzyme and its activity. The activity of the purified protein was determined through different parameters, including various ranges of temperature, pH, and time. A linear increase in the enzyme activity was observed upon the increase in MTase concentration from 0–5 µM (Figure 2A). Further, enzyme activity was studied in the pH range from 6 to 12, leading to a bell-shaped curve with an optimal pH of 8.0 (Figure 2B). Additionally, the optimal temperature of the enzyme activity was found to be 37 ◦C (Figure 2C), above which the enzyme became denatured. Further, through the time-course assay, the optimal enzyme activity was found at 150 min, after which it plateaued (Figure 2D). For all future studies, the concentration of the enzyme used was 0.675 µM, while the concentration of GTP used was 0.4 mM. The graphs were plotted in terms of S-adenosyl homocysteine (SAH) concentration, determined using the standard curve. All the reactions were performed in triplicate, and the readings were subtracted from those of the no enzyme control. Each data point in the graph represents the mean value, and the error bars indicate the standard deviation.

### zyme control. Each data point in the graph represents the mean value, and the error bars indicate the standard deviation. 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 k<sup>m</sup> value for GTP, which was found to be 0.387 mM (Figure 3A). The k<sup>m</sup> value was also determined using a Lineweaver–Burke plot produced by GraphPad Prism 9. The straightline equation for the plot, Y = 0.0003207 × X + 0.0008274, was used to calculate the k<sup>m</sup> and Vmax values (Figure 3B). The calculated k<sup>m</sup> and Vmax values from the Lineweaver–Burke plot were found to be 0.387 mM and 120,496, respectively.

**Figure 2. Effect of various parameters on MTase activity:** The MTase activity was optimised by adjusting various parameters. (**A**) MTase activity at varying enzyme concentrations. (**B**) Enzyme activity at different pH values. (**C**) Enzyme activity at different temperatures (**D**). Enzyme activity at various time points. The observations are made from experiments performed in triplicate. Each data point on the graph represents the mean value, and the error bars indicate the standard deviation. **Figure 2. Effect of various parameters on MTase activity:** The MTase activity was optimised by adjusting various parameters. (**A**) MTase activity at varying enzyme concentrations. (**B**) Enzyme activity at different pH values. (**C**) Enzyme activity at different temperatures. (**D**) Enzyme activity at various time points. The observations are made from experiments performed in triplicate. Each data point on the graph represents the mean value, and the error bars indicate the standard deviation.
