(BIO) Enzyme Kinetics and Cellular Metabolism Flashcards
Km
Michaelis-Menten constant
Represent the substrate concentration for the reaction rate to reach 1/2 Vmax
Vmax
The maximum reaction rate reached by the system, when all active sites are occupied
Competitive Inhibitors
Compete with the substrate to bind to the active site of the enzyme
Increase substrate concentration will reduce the chance of the inhibitor binding to the enzyme
Can be outcompeted by increase in substrate concentration
Noncompetitive Inhibitors
Bind the enzyme at a site other than the active site
This can occur with or without the substrate present
Increase substrate concentration will NOT relive the inhibition, since the inhibitor binds the enzyme-substrate complex (ES) just as it binds the enzyme
Uncompetitive Inhibitors
Bind only to the ES complex
This reduction in the effective ES concentration:
Increases the enzyme’s apparent affinity for the substrate
Decreases the maximum activity of the enzyme
Kcat
Aka Turnover #
Denotes the number of substrate molecules converted per enzyme molecule per second
Provides a direct measure of the catalytic production of product under optimum conditions
Enzyme Efficiency
Reflected by the quantity of Kcat/Km
(Kcat)/(Km) > 1 when:
- Turnover number is high (Large Kcat)
2. Enzyme has high affinity for substrate (low Km)
Michaelis-Menten Curve
X axis = Substrate concentration [S]
Y axis = Reaction velocity (v)
@ 1/2 Vmax, substrate concentration = Km
Competitive Inhibition = just below normal curve but eventually reaches same Vmax, lower Km but same 1/2Vmax
Noncompetitive Inhibition = Vmax is 1/2Vmax of normal curve, starts out the same as competitive inhibition, lowest Km & 1/2Vmax
Lineweaver- Burk Plots
X-axis = 1/[S] Y-axis = 1/Vo Slope = Km/Vmax
Competitive = same 1/Vmax, diff -1/km Noncompetitive = diff 1/Vmax, same -1/km Uncompetitive = diff 1/Vmax, diff -1/km
Glycolysis
Step 3 = committed step, rate limiting step
F6P –> (PFK + ATP) –> F16BP
PFK
Enzyme in Step 3 for F6P –> F16BP
Regulated by: (+) AMP (+) F26BP --> closely related to insulin & glucagon; when insulin is active F26BP is produced (-) ATP (-) Citrate
Regulation Steps of Glycolysis
Highly EXERgonic steps which make reverse rxn impossible!
Step 1:Glucose –> (Hexokinase + ATP) –> G6P
Step 3: F6P –> (PFK + ATP) –> F16BP
Step 10: PEP –> (Pyruvate kinase + ATP) –> Pyruvate
Glycolysis Yields
Net 2 ATP
4 ATP + consumes 2 ATP
Glycolysis occurs in the
Cytosol and is ANaerobic
What reaction bridges the gap from Glycolysis to the Citric Acid Cycle?
Pyruvate Decarboxylation (catalyzed by pyruvate dehydrogenase complex)
Regulation of Glycolysis Conceptually
- Regulation of glucose entrance into the cell
Increase in blood glucose increases insulin, leading to localization of GLUT4 at the cell surface - Regulation of the enzymes involved in the metabolic pathway (Steps 1, 3, and 10)
Irreversible steps and have large negative deltaG˚ values - Regulation in response to concentrations of certain effectors
Increase in ATP concentration signals that energy produced is greater than energy consumed, so the body can downregulate glycolysis and upregulate glycogenesis
Increase in Citrate concentration also means that energy levels are high and intermediates are abundant
1 Glucose molecule produces
2 Pyruvates
2 Net ATP
How many rounds of the Citric Acid Cycle result from 1 round of Glycolysis?
1 Glucose –> 2 Acetyl CoA = 2 turns of the cycle
All C, H and O from Pyruvate end up as CO2 and H2O
What is the net gain of ATP from the Citric Acid Cycle?
Per 1 Glucose:
6 NADH
2 ATP
2 FADH2
Where in the cell does the Citric Acid Cycle occur?
Mitochondrial Matrix
The electrons used in the ETC are produced by the TCA via the reduction of…
NAD and FAD
Citric Acid Cycle Regulation (via PDC: pyruvate dehydrogenase complex)
Activated by:
NAD+
CoA
AMP
Inhibited by plenty of fuel:
NADH
Acetyl-CoA
ATP
TCA Regulation (Main Mechanisms)
- Substrate (Oxaloacetate and Acetyl-CoA) concentration
- Product (NADH) concentration
- Allosteric activation
- Competitive feedback
TCA Regulation: Substrate concentration
Oxaloacetate and Acetyl-CoA concentration
TCA Regulation: Product (NADH) concentration
NADH inhibits PDC and other regulatory steps
TCA Regulation: Allosteric activation
ADP is an allosteric activator of TCA
TCA Regulation: Competitive feedback
Succinyl-CoA and intermediate, competes with acetyl-CoA to bind Citrate Synthesis
ETC Goal
Create an H+ gradient
ETC Gradient must be present in order to
Form ATP using the enzyme ATP Synthase
Because the binding of ADP and (PO4)3- and the subsequent release of ATP from the enzyme actually requires energy
Reduction Potential (E˚)
The tendency to accept electrons
Electrons flow spontaneously from
Lower E˚ to Higher E˚
DeltaG = -nF (deltaE)
ETC yield of ATP
2 NADH from Glycolysis 2 NADH from PDC 6 NADH from TCA = 10 NADH x 2.5 =25 ATP
2 FADH2 from TCA
= 2 FADH2 x 1.5
= 3 ATP
Total ATP = 28 ATP
Where does ETC occur?
Along the INNER Mito Matrix
Infants have brown fat cells which express a protein that acts to dissipate the proton gradient, yet allows O2 consumption to continue.
What effect might this have on ATP generation?
Why might this mechanism have evolved?
Dissipating the proton gradient would make the ETC much LESS efficient. ATP generation would decrease, while more energy would be lost as waste
Potential evolutionary explanation: brown fat aids in thermogenesis
What is the final electron acceptor of ETC?
Oxygen
Without O2, cells must rely on anaerobic respiration (glycolysis and fermentation)
Anerobic Respiration
Glycolysis and Fermentation
Fermentation
Converts NADH to NAD+
