Assay of EPSPS EPSPS was assayed by following the disappearance of PEP. Reaction mixtures (Reaction A: total volume 0.5mL ) contained 3mM PEP, 3mMS3P,50mM Pipes buffer ( pH 6.6) and enzyme sample (0.1mL). After incubation at 37C for 15 min, the reaction was stopped by incubation at 100C for 3min. Under these conditions it has been shown that the rate of disappearance of PEP is linear during the 15 min assay. The amount of PEP remaining was then determined in a spectrophotometer set at 340nm using a cuvette with a 1cm lightpath and a coupled enzyme reaction (as shown below). 0.05mL of the initial reaction A mixture was added to a mixture 4 (0.85 mL) containing pyruvate kinase, lactate dehydrogenase and ADP and the spectrophotometer was set to zero. 0.1mL of 2mMNADH was then added to complete Reaction B and after 2 minutes the final A340 was measured. The resulting change in A340 when the 0.1mL enzyme sample was taken from 82mL of crude pea extract was 0.738. Take the value of 340 for NADH to be 6220M1 cm1. PEP Pyruvate Lactate 2. Calculate the amount of PEP present in Reaction A at the start of the assay reaction in nmols. 3. Calculate the amount of PEP present in Reaction A after the reaction with pea extract in nmol. Remember that only 0.05mL of the total volume of reaction A (0.5mL) was used. 4. Calculate the change in amount of PEP per minute in reaction A, and hence calculate the number of units of EPSPS present in the 82mL of initial cell extract. [Where 1 unit is defined as the amount of enzyme that catalyses the reaction of 1 nmol PEP per minute.] Enter this value in the table below