Metabolic ReactionsEnzymologyCatabolism
Litho/PhototrophyAnabolism
Microbial Metabolism
Enzymology• Enzymatic Catalysis• Holoenzymes & Cofactors• Enzyme Activity
– Environmental Conditions– Substrate Concentration (Enzyme Kinetics)– Enzyme Content– Competitive & Noncompetitive Inhibitors– Allosteric Regulation (negative & positive)– Reversible Covalent Modification
• Metabolic Pathways– Basic Configurations (Types)– Pathway Activity
• Feedback Inhibition• Feed-Forward Activation
– Amphibolic Pathways
Enzymatic Catalysis• Enzyme: protein catalyst; increases rate of conversion
of substrate to product.• Active (catalytic) site has an affinity for substrate(s).• Substrate can fit the active site like a lock-and-key.• Active sites of others are induced to fit the substrate.• Formation of an enzyme-substrate complex stabilizes
the transition-state between substrate to product.
• The active site reduces the reaction activation energy.• Even thermodynamically spontaneous reactions can
have slow rates without an enzyme to act as catalyst.
= transition-state
No enzyme
With enzyme
Enzymatic Catalysis
Holoenzymes and Cofactors• Some enzymes (holoenzymes) require a cofactor and
apoenzyme protein component for activity.• Cofactors are often involved in the redox reactions.• Cofactor Types:
– Prosthetic groups (permanently bound to apoenzyme).– Coenzyme (loosely binds to apoenzyme as needed).
Enzyme Activity• Environmental factors influence enzyme activity (temperature, pH, water
activity, barometric pressure).• Extreme conditions denature proteins.
Enzyme Activity• Increased substrate concentration can increase activity to some maximum velocity (Vmax); active site becomes saturated.• Vmax can increase by expressing more enzyme; increase enzyme content• Michaelis constant (Km) is the concentration at ½Vmax; reflects enzyme affinity for substrate; lower values mean higher affinity.
Enzyme Activity• Enzyme activity may change due to inhibitor molecules.• Competitive inhibitors occupy the active site• Noncompetitive inhibitors bind to an allosteric (regulatory) site; it’s separate from
the active site yet distorts the protein so the active site no longer binds the substrate.
Enzyme Activity• Enzyme activity can change due to activator molecules. • Like noncompetitive inhibitors, activators bind to an allosteric (regulatory)
site; however the effect is enhanced binding of substrate.• Generally, activators and noncompetitive inhibitors of this type are called,
effectors.• Enzyme regulation of activity by an effector binding an regulatory site is
referred to as allosteric regulation. This kind of regulation is reversible.
Enzyme Activity• Enzyme activity may
change due to reversible covalent modification.
• Covalent binding of a particular functional group (phospho-, methyl-, adenyl-, etc…) may increase or decrease activity of the target enzyme.
• Modified enzymes may be returned to the original form; hence these are called interconvertible enzymes
• Other enzymes are responsible for the “taking on and off” of the modifying functional group, these are called converter enzymes and may themselves be under allosteric regulation.
Metabolic Pathways • Although we can recognize a substrate and product of
individual enzymatic reactions; metabolic functions are often performed by several enzymatic reactions in a “pathway”.
• Pathways can be linear, branched, cyclic or even spiral.
• Pathway activity is controlled in three ways:– Metabolites and enzymes may be localized in different parts of
the cell; called metabolic channeling.
– The total amount of enzymes in a pathway can vary (gene expression).
– Pathway activity is controlled by critical regulated enzymes. These “pacemaker enzymes” are often the rate-limiting step in the pathway.
Metabolic Pathways
Feedback Inhibition: (“end-product inhibition”)
• rate limiting enzyme is first in pathway and allosteric.
• end-product is a negative effector (inhibitor) of first enzyme
Metabolic Pathways
+Feed Forward Activation:• rate limiting enzyme of a branch point is allosteric. • earlier-substrate is a positive effector (activator) of forward reaction enzyme.
NOTE: the example also illustrates feedback inhibition.
Metabolic Pathways
• Amphibolic Pathways– Catabolic direction– Anabolic direction
• Separate regulatory enzymes each way; function as “check valves” for flow control.
• Other pathway enzymes are reversible; ΔGo’≈0; their equilibrium shifts based on concentration of reactants & products.
• Gycolysis is a good example.
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