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