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Chapter 9
Enzyme Regulation
Metabolic Pathways
• Regulation will depend on ability to alter flux thru the pathway by activation of the rate-limiting enzyme
Common Themes
• Types of regulation depend upon the particular pathway and importance of pathway in the cell/tissue.– Negative feedback– Feed forward
• Tissue specific isozymes
Mechanisms of Regulation
• Regulation by compounds that bind reversibly to the active site
• Regulation by alteration of the active site
• Regulation by changing the concentration of the enzyme
Michaelis-Menton
• Describes response of an enzyme to changes in substrate concentration
• Powerful tool used to study normal and altered enzymes, such as those that produce diseases.
• Like Hendersson-Hasselbach, LIVE IT< LOVE IT< LEARN IT.
Michaelis-Menten-2
The Reaction
The Dreaded Equation
Graph of Michaelis-Menton
Michaelis-Menten-3
• Hyperbolic kinetics, saturation kinetics
• [S]>>Km v=Vmax• [S]=Vmax
v=Vmax/2• [S]<<Km velocity
depends linearly on [S]
Lineweaver-Burk Plots
Warning
• Michaelis-Menten does not describe all enzymes– E.g. glucokinase
• The model can not be used in situations in which [E]>[S]
Hexokinase Isozymes• Catalyze the same reaction BUT
• Different Km for glucose
• Always remember that activity of enzymes will always depend upon the needs of a particular tissue.
Glucose Metabolism
Hexokinase I and Glucokinase
• Hexokinase I RBCsKm= 0.05 mM
• Glucokinase liverKm =5-6 mM
Glucokinase has a Higher Vmax
• Prevents glucose from entering systemic circulation following carb-rich meal; minimizes hyperglycemia
MODY2
• Maturity Onset Diabetes of the Young Type 2– Defect in pancreatic
glucokinase– Associated with a reduction
of level of insulin release for certain level of glucose
Velocity and Enzyme Concentration
• Rate is directly proportional to the concentration of enzyme.
Inhibition within the Active Site
• Inhibitors decrease rate of reactions
• Inhibitors may be reversible or irreversible– Refer to Ch 8 on mechanism
based inhibitors (irreversible)
Competitive Inhibition• Inhibitor COMPETES with
substrate for the active site
Competitive Inhibition
• Inhibitor can be diluted by increasing [S]– Vmax unchanged
• Apparent Km increases– We needed to raise [S] to
outcompete the inhibitor & saturate the enzyme
Graphical Depiction of Competitive Inhibition
Competitive Inhibition in Real Life
• Al Martini and his alcohol dehydrogenase (ADH)– Ethanol + NAD+ Acetaldehyde
+ NADH + H+
– As more and more alcohol is being oxidized, the NADH/NAD+ ratio increases
NADH Inhibition of Enzymes
– NADH competes with NAD+, thereby inhibiting ADH• Ethanol clearance from blood slows
– NADH also inhibits enzymes involved in FA oxidation• Contributes to alcoholic fatty liver
Competitive Inhibition
• Zocor and Lipitor inhibit HMG CoA reductase– Inhibits de novo cholesterol
synthesis• Use of ethanol to treat
ethylene glycol and methanol poisoning
Noncompetitive Inhibition
Binds to site other than active site or
Does not compete with a substrate for binding site
Noncompetitive Inhibition
• Essentially no competition• Decreases available/effective
enzyme concentration– Vmax decreases– Km unchanged if pure
noncompetitive
Graphical Depiction of Noncompetitive
Inhibition
Uncompetitive Inhibition
• Reduce effective enzyme concentration– Vmax decreases
• Inhibitor binds only ES– Km decreases
Regulation through Conformational Changes
• Do not affect [E]• Respond quickly• Responsible for moment to moment
regulation of activity• Mechanisms:
– Allosteric– Reversible covalent modification– Control proteins
Allosteric Regulation
• Regulation through binding of allosteric effectors– Bind to site separate from
catalytic site (allosteric site)• Positive effectors activate
enzyme• Negative effectors inhibit
activity
Allosteric Inhibition
• Allosteric modulators can indirectly alter the configuration of the active site, rendering the enzyme inactive
• Noncompetitive inhibitors work by this mechanism
Allosteric Activation
• An enzyme site may be activated sterically by an allosteric modulator
Properties of Allosteric Enzymes
• Oligomeric = 2 or more subunits
• Exist in 2 conformational states (R or T)
• Exhibit cooperativity– Display sigmoid
saturation curves
Activators and Inhibitors of Allosteric Enzymes
Activator binds R state
Inhibitor binds T state
Covalent Modification
• MAJOR method for rapid and transient regulation of enzyme activity
• Human genome encodes for > 1,000 different protein kinases– I guess protein
phosphorylation is important!!
Phosphorylation & Dephosphorylation
• ON and OFF Switch
• Addition or removal of a phosphate group
– Which amino acid residues get phosphorylated?
Serine, threonine or tyrosine
Protein kinases and phosphatases
• Kinases add a phosphate
• Phosphatases remove a phosphate
Muscle Glycogen Phosphorylase
Rate-limiting step in pathway of glycogen breakdown [glycogen glucose 1-P]
Regulated by allosteric activator AMP AND
by phosphorylation
Protein-protein Interactions
• Modulator proteins change shape of catalytic site or blocks the site
• Calcium-calmodulin – Regulates a large # of
proteins• G-proteins
Calcium-Calmodulin
Neural impulse triggers calcium release from SR
Calcium binds calmodulin subunit of muscle glycogen phosphorylase kinase
Activated kinase then phosphorylates glycogen phosphorylase
Proteolytic Cleavage
• Proenzymes = enzymes that must undergo proteolytic cleavage to be active
• IRREVERSIBLE form of regulation• Zymogens = precursor proteins of
proteases– Chymotrypsinogen, trypsinogen– Fibrinogen, prothrombin
Common Themes Concerning the
Regulation of Metabolic Pathways
• What are pathways?• Regulation occurs at Rate-
Limiting Steps
Themes -2
• Regulation matches function • Feedback Regulation- Negative
Feedback• Feed-Forward Regulation• Counter- regulation
– Keep opposing pathways separate• Compartmentation
– Special needs– Limitation of substrate