Using the appropriate graph and table above, explain what the N426S mutation appears to be doing to the enzyme’s function. Discuss the kinetic parameter changes and their meaning in this context, not the structure of the enzyme, which was not given to you. The image consists of three line graphs labeled d, e, and f, each displaying the relationship between substrate concentration and relative velocity (v/v).### Graph d: [Citrate] vs. Relative Velocity- **Axes:** - X-axis: [Citrate] (mM), ranging from 0 to 4 mM. - Y-axis: Relative velocity, v/v, ranging from 0 to 1.0.- **Curves:** - Four curves are shown using different colors and symbols (e.g., red squares, green triangles, blue triangles, black circles). - The curves depict how citrate concentration affects the relative velocity of a reaction. - As citrate concentration increases, relative velocity generally decreases, particularly in three curves, while the red curve remains relatively constant.### Graph e: [ATP] vs. Relative Velocity- **Axes:** - X-axis: [ATP] (mM), spanning from 0 to 3 mM. - Y-axis: Relative velocity, v/v, from 0 to 1.0.- **Curves:** - Four curves represented with distinct colors and markers. - The relative velocity initially increases with ATP concentration, peaking around 0.5 - 1 mM, then declines as ATP concentration continues to rise.### Graph f: [F6P] vs. Relative Velocity- **Axes:** - X-axis: [F6P] (mM), ranging from 0 to 4 mM. - Y-axis: Relative velocity, v/v, ascending from 0 to 1.0.- **Curves:** - Four colored curves indicate how the relative velocity varies with fructose 6-phosphate concentration. - Relative velocity increases with increasing [F6P], suggesting a sigmoidal relationship.### General Observations:Each graph provides insights into how different substrate concentrations ([Citrate], [ATP], [F6P]) influence the relative velocity of biochemical reactions. The error bars indicate variability and potential experimental error. ## Enzyme Assay Data for PFK-1 Protein Variants### Table: Kinetic Parameters| **PFK-1 protein** | **Vmax** | **Km fructose 6-phosphate** | **Ki ATP** | **Ki citrate** ||------------------|----------|-----------------------------|------------|---------------|| **Wild type** | 59.27 | 0.83 mM | 0.96 | 0.4 || **R48C** | 58.19 | 0.84 mM | 1.19 | >4 || **N426S** | 67.41 | 0.82 mM | >3 | 0.31 || **D546N** | 30.6 | 2.04 mM | 0.68 | 1.4 |### DescriptionThe data in the table are derived from the enzyme assays represented in the graphs. The **red squares** denote the R48C variant, **blue triangles** denote N426S, **green upside down triangles** denote D546N, and **black circles** denote the wild type PFK-1. Fructose 6-phosphate and ATP, the substrates, are present in all assays.- **Graph d:** Measures the velocity of PFK-1 with respect to varying **citrate concentration**.- **Graph e:** Measures the velocity of PFK-1 with respect to varying **ATP concentration**.- **Graph f:** Measures the velocity of PFK-1 with respect to varying **F6P concentration**.These kinetic parameters and graphs help in understanding the differences in enzyme activity and substrate affinity among the PFK-1 protein variants.

Regulatory enzymes exhibit increased or decreased catalytic activity in response to specific signals. Activities of regulatory enzymes can be modulated by reversible covalent or non-covalent binding by regulatory compounds. According to Michaelis-Menten Kinetics, when the rate or velocity of an enzyme catalyzed reaction is measured at varying substrate concentrations, the rate depends on the substrate concentrations [S]. At a relatively low concentration of substrate, initial velocity (V0) increases almost linearly with an increase in substrate concentration. The reaction reaches a maximum velocity (Vmax) with an increase in substrate concentration, and it does not increase any further by increasing in substrate concentration. Concentration of substrate at which the reaction velocity reaches half of the maximum velocity is called Michaelis constant or Km. PFK1 or phosphofructokinase 1 is the most important allosteric enzyme which takes part in glycolysis. It is inhibited by the increased concentration of ATP. ATP and citrate act as a inhibitory or negative modulator which binds at the allosteric site of PFK1 enzyme and inhibit the enzyme activity. So, at low concentration of ATP, the reaction rate of the enzyme PFK1 is higher, and the Km is lower. But with increasing ATP concentration, Km is also raised for the substrate. Hence the velocity of the enzyme activity declines, due to inhibition by ATP molecules resulting in low reaction rate. Fructose-6-phosphate stimulates the activity of wild type phosphofructokinase 1. In case of N426S mutant, the Vmax has increased but the Kmfructose6P is the same as wild type and KATP has increased and Kcitrate is almost the same as wild type. This means the mutant has caused changes in the active site and the regulatory site of the enzyme which contains different amino acids. This has lead to altered substrate-enzyme and ligand-receptor binding which is directly altering the Km and Vmax of the enzyme.