experiment 10: enzyme kinetics of LDH

Question 1

explain why it is important for scientists to study enzyme kinetics

Answer 1

• allows for comparison of similar reactions to determine if two enzymes use similar mechanisms
• can determine order of substrate addition and product release
• helps evaluate the efficiency of pharmaceutical inhibitors
• can determine efficiency of an enzyme by measuring turnover rate

Question 2

what factors can influence enzyme catalyzed reactions?

Answer 2

temperature: phosphatase experiment 4-55 degrees celsius
pH: pH5 vs pH8
ionic strength: salt
cofactor concentration: [ATP] [Mg2+]
inhibitor/activator concentration

Question 3

what is the difference between a continuous time course assay and a fixed-time course assay?

Answer 3

continuous time: continuous recording of an assay over time to make a trendline

fixed-time: records an assay at certain time points, a trendline is not apparent

Question 4

under what conditions would a fixed-time course assay be inappropiate?

Answer 4

if you do not know concentration range of salt over which a protein of interest will elute, also bad for doing enzyme kinetic michaelis-menten plots

Question 5

what are the assumptions of the Michaelis-Menten equation?

Answer 5

[E] remains constant, enzyme not consumed
[ES] formation is rapid and constant (equilibrium)
there is only one substrate
[S][cofactor]&raquo_space;»> [E]
reaction proceeds in one direction
t=0, [P]=0, [S]~[S]initial
product converted back into substrate

Question 6

define Vmax, Km, and Kcat

Answer 6

-Vmax: rate of reaction, measure of how fast an enzyme can catalyze a reaction, measured with a specific enzyme concentration
-Km: concentration expressed in M, measure of substrate concentration needed for effective catalysis (high Km=high [S] needed) (low Km=high affinity for substrate)
-Kcat: measure of how many substrate molecules are converted per enzyme molecule per second

Question 7

which Km value is probably more accurate when using a Michaelis-Menten or Lineweaver-Burke plot and why?

Answer 7

Lineweaver-Burke plot gives an equation of straight line to linearize the data from M-M plot. Km determined from x-intercept and is more convenient and accurate.

M-M plot obtained from extrapolation from curve at 1/2Vmax, difficult to obtain Km from this curve

Question 8

what is the physiological role of LDH enzyme and the NAD+ cofactor?

Answer 8

LDH: converts pyruvate to lactate or back by oxidation of NADH to NAD+ or reduction of NAD+ to NADH

NADH absorbance value measured at 340 nm

NAD+ is a proton acceptor

Question 9

under what condition(s) is the conversion of pyruvate to lactate reversible?

Answer 9

under aerobic conditions (when oxygen is present) NADH would be converted to NAD+ by the electron transport chain. pyruvate can then be converted to acetyl-coA and through go through the TCA cycle

Question 10

be able to determine Vmax, Km, and Kcat from M-M or Lineweaver-Burke plot

Answer 10

• M-M: 1/2Vmax and then interpolate to get Km
• Lineweaver-Burke: y-intercept is 1/Vmax, x-intercept is -1/Km
• kcat=Vmax/[E] where [E] is given or enzyme molecular weight given