Metabolic Reactions Enzymology Catabolism Litho/Phototrophy Anabolism 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 (V max ); active site becomes saturated. V max can increase by expressing more enzyme; increase enzyme content Michaelis constant (K m ) is the concentration at ½V max ; 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; ΔG o ’≈0; their equilibrium shifts based on concentration of reactants & products. Gycolysis is a good example.