F23 Exp 10 Lab Report(1)

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BIOL 3380 Experiment 10 - Lab Report Enzyme Kinetics of LDH Name: Circle your lab section: T-AM W-AM R-AM F-AM T–PM W-PM R-PM F-PM Instructor: Jing Pan Graduate TA: Date: 11/25/2023 Partner:

Experiment 10 – Lab Report Enzyme Kinetics of LDH Edit this word document by inserting your answers after each question/space. Please denote your answers by using bold , italic s, or colored font. Do NOT renumber/reorganize the questions. “Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so). Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate. All figures/graphs/tables must include appropriate titles, legends, and labeled/ scaled axes. 1. (3pts) Present a table showing initial velocity data (  A 340nm /min,  NADH[nM]/min, and ∆NADH[nmol]/min) as a function of LDH concentration. 2. (3pts) Present a graph of  NADH[nmol]/min as a function of LDH concentration.

3. (3pts) Present a table showing initial velocity data (  A 340nm /min,  NADH[nM]/min, and ∆NADH[nmol]/min) as a function of lactate concentration. 4. (5pts) Present a graph of  NADH[nmole]/min as a function of lactate concentration (Michaelis-Menten plot).

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5. (5pts) Convert the Michaelis-Menten plot into a Lineweaver-Burke plot. 6. (4pts) What is the V max as determined by the Michaelis-Menton plot? What is the V max as determined by the Lineweaver-Burke plot? Why is there a difference (if any)? Show your work. The vmax on the Michaelis-Menton plot is 57.92 nmol/min and the vmax on the Lineweaver- Burke plot is 0.0667 nmol/min. The Lineweaver-Burke plot, which is more accurate than the Michaelis-Menten plot, extrapolates data by using the average to obtain the Vmax, which accounts for the difference in Vmax. Using the Lineweaver-Burke plot is more practical and

believable than using the Michaelis-Menten plot, which would need us to use an infinite amount of substrate concentration to determine the genuine Vmax. Furthermore, the Lineweaver-Burke plot eliminates the need for guesswork by utilizing the y-mx + b equation to compute and determine the precise Vmax. 7. (4pts) What is the K m as determined by the Michaelis-Menten plot? What is the K m as determined by the Lineweaver-Burke plot? Why is there a difference (if any)? Show your work. The Km from the Michaelis-Menten plot is about [Lactate] = 1.8 nm whereas the Km from the LB plot is [Lactate] = 2.39nm. There is a discrepancy in Km because the Lineweaver-Burke plot computes the precise Km number by utilizing the y-mx + b equation and a trendline to establish an average of all the data points. The Michaelis-Menten plot, on the other hand, asks us to calculate the concentration and half of the Vmax. 8. (3pts) Using the data from the Lineweaver-Burke plot, what is the calculated turnover rate for an LDH enzyme molecule? An LDH enzyme molecule is composed of 331 amino acids. An average amino acid weighs 110 Daltons. Show your work. (1 Dalton = 1g/mole)

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