effect of environment on enzyme activity substrate concentration ph temperature

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

Stage 2

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

Stage 3

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