Effect of environment on enzyme activity Substrate concentration pH Temperature

Substrate concentration Enzyme activity increases with increasing substrate concentrations At a certain concentration the enzyme will be saturated and operate at maximum velocity = Vmax

Substrate concentration Plot of velocity (activity) vs. substrate concentration results in a hyperbola

Michaelis constant (Km) Used to measure the affinity of an enzyme for its substrate Km = substrate concentration required to achieve half maximal velocity

Effect of pH and temperature on enzyme activity Enzymes are most active at optimum pH and temperatures Deviations from the optima can slow activity and damage the enzyme

Effect of pH and temperature on enzyme activity Loss of activity due to extreme pH, temperature or other factors is known to as denaturation Temperature and pH optima of microorganism’s enzymes usually reflect the microorganism’s environment

Enzyme inhibition Many poisons and antimicrobial agents are enzyme inhibitors Can be accomplished by competitive or noncompetitive inhibitors Competitive inhibitors - compete with substrate for the active site Noncompetitive inhibitors - bind at another location

Competitive inhibitors Usually resemble the substrate but cannot be converted to products

Noncompetitive inhibitors Bind to the enzyme at some location other than the active site Do not compete with substrate for the active site Binding alters enzyme shape and slows or inactivates the enzyme Heavy metals often act as noncompetitive inhibitors (e.g. Mercury)

Metabolic regulation Important to conserve energy and resources Cell must be able to respond to changes in the environment Changes in available nutrients will result in changes in metabolic pathways

Metabolic regulation Metabolic channeling Stimulation or inhibition of enzyme activity Transcriptional regulation of enzyme production

Allosteric enzymes Activity of enzymes are altered by small molecules known as effectors or modulators Effectors bind reversibly and noncovalently to the regulatory site Binding alters the conformation of the enzyme

Allosteric enzymes Positive effectors increase activity Negative effectors decrease activity

ACTase regulation Regulation of aspartate carbamyltransferase is a well studied example of allosteric regulation CTP inhibits activity and ATP stimulates activity

ACTase regulation Binding of effectors cause conformational changes that result in more or less active forms of the enzyme

ACTase regulation Binding of substrate also increases enzyme activity (more than one active site) Velocity vs. substrate curve is sigmoid

Covalent modification of enzymes Attachment of group to enzyme can result in stimulation or inhibition of activity Attachment is covalent and reversible

Covalent modification of enzymes Attachment of phosphate groups often used to regulate enzyme activity Other groups can also be used to regulate enzyme activity

Feedback inhibition Metabolic pathways contain at least one pacemaker enzyme Usually catalyzes the first reaction in the pathway Activity of the enzyme determines the activity of the entire pathway

Feedback inhibition Feedback inhibition occurs when the end product interacts with the pacemaker enzyme to inhibit its activity Branching pathways regulate enzymes at branch points

Overview of metabolism Metabolism = the total of all chemical reactions occurring within the cell Catabolism = the breaking down of complex molecules into simple molecules with the release of energy Anabolism = the synthesis of complex molecules from simple molecules with the use of energy

Sources of energy Microorganisms use one of three sources of energy Phototrophs - radiant energy of the sun Chemoorganotrophs - oxidation of organic molecules Chemolithotrophs - oxidation of inorganic molecules

Electron acceptors Chemotrophs vary regarding their final electron acceptors Fermentation - no exogenous electron acceptor is required Aerobic respiration - oxygen is the final electron acceptor Anaerobic respiration - another inorganic molecule is acceptor

Electron acceptors Chemolithotrophs can use oxygen or another inorganic molecule as the final electron acceptor

The three stages of catabolism Catabolism can be broken down into three stages Stage 1 Larger molecules (proteins, polysaccharides, lipids) are broken down into their constituents Little or no energy is generated

The three stages of catabolism Amino acids, monosaccharides, fatty acids, glycerol and other products degraded to a few simpler products Can operate aerobically or anaerobically Generates some ATP and NADH or FADH

The three stages of catabolism Nutrient carbon is fed into the tricarboxylic pathway and oxidized to CO2 ATP, NADH and FADH produced ATP generated from oxidation of NADH and FADH in electron transport chain

Amphibolic pathways Pathways that can function both catabolically and anabolically Glycolysis and the tricarboxylic acid cycle are two of the most important amphibolic pathways Most reactions are reversible

The glycolytic pathway/glycolysis Also known as the Embden-Meyerhof pathway Most common pathway of degradation of glucose to pyruvate Found in all major groups of microorganisms Can function aerobically or anaerobically

The glycolytic pathway/glycolysis Occurs in 2 stages The six-carbon stage Glucose is phosphorylated 2x and converted to fructose-1,6-bisphosphate Other sugars converted to glucose-6-phosphate or fructose-6-phosphate and fed into pathway

The glycolytic pathway/glycolysis The six-carbon stage Does not yield energy Uses 2 ATPs Serves to “prime the pump”

The glycolytic pathway/glycolysis The three-carbon stage Fructose-1,6-bisphosphate split in half by fructose-1,6-bisphosphate aldolase Yields glyceraldehyde-3-phosphate and dihydroxyacetone phosphate

The glycolytic pathway/glycolysis The three-carbon stage Dihydroxyacetone phosphate readily converts to glyceraldhyde-3-phosphate Fructose-1,6-bisphosphate  2 glyceraldehyde-3-phosphate

The glycolytic pathway/glycolysis The three-carbon stage Glyceraldehyde-3-phosphate converted into pyruvate in 5 steps Oxidized by NAD+ and a phosphate is added  1,3-bisphosphoglycerate Phosphate on carbon 1 donated to ADP to form ATP Substrate-level phosphorylation

The glycolytic pathway/glycolysis The three-carbon stage 3-phosphoglycerate shifted to carbon 2  2-phosphoglycerate Dehydration results in high energy phosphate bond in phosphoenolpyruvate Phosphate transferred to ADP to form ATP (substrate-level phosphorylation)

The glycolytic pathway/glycolysis Glucose  2 pyruvates + ATP +NADH 2 ATP used in six-carbon stage 4 ATP + 2 NADH formed in three-carbon stage

The glycolytic pathway/glycolysis Glucose + 2ADP + Pi + 2NAD+  2 pyruvate + 2ATP + 2NADH