Microbial Metabolism Metabolic Reactions Enzymology Catabolism LithoPhototrophy Anabolism

Microbial Metabolism Metabolic Reactions Enzymology Catabolism Litho/Phototrophy Anabolism

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.

Enzymatic Catalysis • 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

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, p. H, 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.