1. Enzyme X has a molecular weight of 35,000 Da. When this enzyme is assayed forenzyme activity, the corresponding line of the double reciprocal plot has the equation y =75,253X + 102,392. The substrate concentration was in mM and the velocity wasmeasured in mM min". From these data calculate the following:a. KM and Vmaxb. kcat if the enzyme concentration was 2 mg/mL.2. When you run enzyme X from question one on an SDS-PAGE you find that, afterCoomassie staining, there is only one band on the gel that runs just below the 20,000Dalton molecular weight marker. What does this result tell you about the nature of thequaternary structure of your enzyme?

Enzyme kinetics is used to study the rates of enzyme catalysed biochemical reactions. In this study we measured the effect of substrate concentration on the rate of biochemical reaction using constant enzyme concentration. We can observe the enzyme kinetics by Michaelis-Menten kinetics, which explains the velocity of enzyme catalysed reactions. Lineweaver-Burke plot is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics, in which 1/S vs 1/V plotted to obtain slope and intercept. It is also called double reciprocal plot. Further, we can calculate the values of Vmax and Km from intercept and slopes values, respectively. Lineweaver–Burk plot is correct only when the enzyme kinetics follows second-order kinetics. SDS-PAGE is a method of separation of proteins on the basis of its molecular weight. SDS is ionic detergent which denatures (breaks the quaternary structure of protein) and binds to proteins to make them uniformly negatively charged. So that proteins will be separated solely on the basis of polypeptide length as all protein molecules have same charge. Thus, influence of protein structure and charge will be completely ruled out by the use of SDS. We can observe the size by running the marker proteins in one well. Quaternary structure of the protein forms by the association of several polypeptide chains into a close packed arrangement (like hemoglobin is compose of four polypeptide chains). In which each polypeptide chain will have its own primary, secondary and tertiary structure. In a quaternary structure of protein, its subunits are connected by hydrogen bonds and van der Waals forces via nonpolar side chains of amino acids.1. a. From the given double reciprocal plot equation, the slope and intercept of the graph can be obtained y = m × x + b (y= 75,253 X + 102,392) b (intercept) = 102,392, and m (slope) = 75,253 If we compare these values with Lineweaver-Burke equation 1/Vo = (Km /Vmax) × (1/ S) + 1/Vmax intercept will be, b = 1/Vmax = 102,392 So, Vmax = 9.76 × 10-6 mM/min Now, slope will be, m = Km/Vmax = 75,253 Km = 75,253 × 9.76 × 10-6 = 0.7344 mM So, Km = 0.7344 mM Therefore, Vmax is 9.76 × 10-6 mM/min, and Km is 0.7344 mM, 1. b. For the given enzyme X, molecular weight = 35 kDa , and 1 kDa = ~ 1000 gm/mole Molecular weight of enzyme X = 35000gm/mole So, concentration of enzyme X = 2mg/ml = (2mg/ml) / (35000gm/mole) = (2 × 10-3 × 103) / 35000 mole/L = 0.05714 mM kcat = (Vmax) / (enzyme concentration) kcat = (9.76 × 10-6 mM/min) / 0.057 mM = 171.23 × 10-6 /min = 1.71 × 10-4 /min Thus, kcat is 1.71 × 10-4 /min.2. Enzyme X has molecular weight of 35 kDa. While it gives single band on the SDS-PAGE with molecular weight of <20,00 kDa (~15.5 kDa) as observed by coomassie stain. Thus, this indicates that enzyme X is a dimeric protein which losses its quaternary structure with effect of SDS. SDS is present in loading buffer, and running buffer. SDS is a ionic detergent with a strong protein-denaturing effect and binds to the polypeptide chain with constant molar ratio.