Biochemistry Exam 2

How does an enzyme play a role in the energy barrier

By the interactions within the active site that help stabilize the transition state (enough energy required)

Increases rate not gibbs or equilibrium

For a reaction to occur what 3 conditions are required?

1. Sufficient energy (from collision of molecules)

2. Correct orientation (for necessary interactions)

3. Transition state (not stable)

Gibbs free energy trend?

ΔG < 0 = rxn favored in forward direction

ΔG > 0 = rxn not favored in forward direction

ΔG = 0 = equilibrium

What is gibbs free energy?

Represents the energy available to drive a rxn

Lock and key model

Enzyme shape is complementary to transition state (not substrate)

Allows stability for transition state (lowers activation energy)

What happens if an enzyme is complementary to the substrate

Hard to make a product because lots of activation energy is required.

Factors that influence enzyme activity

1. Temp

2. pH

3. Concentration of substrate, product and/or enzyme

4. Inhibitors and activators

5. Cofactor availability

Changes in enzyme factors affects?

Measured reaction velocity

What does loss of enzyme structure do?

Changes active site, reduces substrate binding, and catalytic activity due to temperature being above the optimal.

What happens to an enzyme if the temperature is too high?

Denaturation - permanent loss of enzyme activity

What does pH changes do?

Changes concentration of hydrogen ions (alters charge of AA side chains in enzyme)

pH of pepsin?

1.5 - 2

pH of trypsin?

7.5 - 8

Reaction velocity

How quickly product forms or substrate disappears

Max velocity (Vmax)

Max rxn velocity of an enzyme catalyzed rxn (enzyme dependent)

Reached when enzyme is saturated with substrate

All active sites are occupied by substrate

Michaelis constant (Km)

Substrates concentration at ½ Vmax

Indicator of an enzyme’s apparent affinity for its substrate

Km trends

Low Km = less substrate required at ½ Vmax

High Km = more substrate required at ½ Vmax

Km is a characteristic of?

A specific enzyme and substrate pair under defined conditions

Michaelis-Mentons equation

Kcat is?

The turnover number (catalytic constant)

= Vmax/[E]total

The larger the number indicates a faster catalytic cycle under saturating substrate conditions

What does Michaelis Menten kinetics provide?

A quantitative framework for understanding enzyme activity, substrate concentrations, Km, and Vmax

What is enzyme inhibition?

Reduction or loss of enzyme activity caused by an inhibitory molecule

Interacts with the enzyme and reduces its ability to catalyze a rxn (does not necessarily change the structure)

Denaturation of an enzyme involves?

Disruption or unfolding of the enzyme structure

Irreversible Inhibition occurs when?

Inhibitor permanently inactivates the enzyme

Reversible Inhibition occurs when?

Inhibitor can be removed and enzyme activity can be recovered

Prevents enzyme from returning to its active form

Irreversible inhibition forms what kind of bonds with enzymes?

Covalent bonds

What are the three types of irreversible inhibitors?

1. Group specific reagents

2. Affinity labels (active site directed reagents)

3. Suicide inhibitors

Types of reversible enzyme inhibition

1. Competitive

2. Uncompetitive

3. Noncompetitive

4. Mixed

Competitive inhibition

Inhibitor is structurally similar to the substrate and acts as a substrate analog

Can be outcompeted by high substrate concentration

Most frequently encountered type of enzyme inhibition

Km increases and Vmax is unchanged

Enzyme inhibtion example

ACE inhibitors - involved in regulation of blood pressure

ACE inhibitors bind to ACE enzyme to reduce the activity

Explain how ACE enzyme works

ACE converts angiotensin I into angiotensin II

Angiotensin II causes blood vessels to constrict and contributes to increased blood pressure

How do ACE inhibitors work?

Binds to ACE and reduces activity

Less angiotensin II produced

Blood pressure decreases

Examples of ACE inhibitors

captopril, enalapril, and lisinopril

Noncompetitive inhibitor

Inhibitor binds to the enzyme at a site other than the active site

Can bind to the free enzyme or the enzyme substrate complex

Reduces the enzyme’s ability to convert substrate into product

Can’t be overcome by increased substrate concentration

Km is unchanged and Vmax decreases

Example of noncompetitive inhibition

Heavy metal inhibition - binds to sulfhydryl group on cysteine and alters enzymes structure

Interferes with enzymes involved in heme synthesis

Uncompetitive inhibition

Inhibitor binds only to the enzyme substrate complex

Inhibitor binds at a site different from the active site

Prevents enzyme substrate complex from forming product efficiently

Increasing substrate concentration does not overcome inhibition

Km and Vmax decrease

Mixed inhibition

Binds at a site different from the active site

Can bind to both the free enzyme and the enzyme substrate complex

Inhibitor has different binding affinities for the free enzyme and enzyme substrate complex

Increasing substrate concentration cannot completely overcome the inhibition

Both Km and Vmax change (binding affinity dependent though)

End product inhibition/ feedback inhibition

A regulatory mechanism in which the final product of a metabolic pathway reduces the activity of an enzyme involved earlier in the pathway

Mechanism of feedback inhibition

End product accumulates and binds to regulatory site on the enzyme

Regulatory site is separate from the active site (allosteric site)

Binding of the end product causes a conformational change in the enzyme and reduces the enzymes activity

Reduced activity slows the pathway and prevents unnecessary production of the end product

Example of end product inhibition

Cholesterol synthesis in the liver is regulated through this mechanism

Cholesterol levels increase = decrease in the activity of enzymes involved in cholesterol synthesis

Limits further cholesterol production when sufficient cholesterol is already availiable

Enzyme catalysis is?

A substrate binds to the enzyme active site, if it is surrounded by specific amino acid side chains

Side chains help position and hold substrate in correct orientation so they can participate in the chemical rxn

General acid base catalysis

Transfer of protons between amino acid side chains in the enzyme and the substrate

AA side chains can donate or accept protons during the rxn

Most common form of enzyme catalysis

Common AAs involve histidine, aspartate, glutamate, lysine, cysteine and tyrosine

Covalent catalysis

Formation of temporary covalent bonds between the enzyme and the substrate

Formation of this covalent intermediate activates the substrate for further rxn

Enzyme is regenerated at the end of the rxn

Metal ion catalysis

Enzyme uses a metal ion to assist in catalysis by stabilizing the transition state, orienting the substrate within the active site, or participate in the transfer of electrons or protons between the enzyme and substrate

Ions include Mg²⁺ , Mn²⁺, Zn²⁺, Fe²⁺, and Cu²⁺

Example of metal ion catalysis

Vitamin B₁₂ contains cobalt which is located in the center of the corrin ring

Cobalt has the ability to form and break bonds with a carbon group

B12 converts methylmalonyl CoA into succinyl CoA

Explain allostery regulation of enzyme activity

Common regulatory mechanism in metabolic enzymes

Regulatory molecule binds away from active site (can cause allosteric activate or end product inhibition of the enzyme)

Explain regulatory subunit regulation of enzyme activity

Binds to specific regulatory molecules that can increase or decrease enzyme activity and or change the enzymes shape

Explain covalent modification regulation of enzyme activity

Enzyme activity can be regulated by adding or removing a chemical group from specific AA residues

Phosphorylation is one of the most common forms (can activate or inhibit activity)

Explain enzymatic degradation regulation of enzyme activity

Increasing enzyme degradation decreases the amount of enzyme availability for catalysis

Ubiquitin can be attached to proteins that are targeted to be degraded by a proteasome

Explain limited proteolysis regulation of enzyme activity

Enzymes synthesized through inactive precursors (zymogens or proenzymes) and goes through this process to active the enzyme through cleaving specific peptide bonds

Example of limited proteolysis

Trypsin is synthesized as inactive zymogen trypsinogen

Trypsinogen is activated by cleavage of a specific peptide bond

Results in an active protease

Common phosphorylation sites?

serine, threonine, and tyrosine

What classic example produces a sigmoidal oxygen binding curve?

Hemoglobin - Binding of one oxygen molecule makes additional oxygen binding more favorable

Allostery example for regulation of enzyme activity

Aspartate transcarbamoylase (ATCase) catalyzes early step in pyrimidine nucleotide biosynthesis

CTP inhibits ATCase

ATP activates ATCase

Regulatory subunits example for regulation of enzyme activity

cAMP is produced from ATP by adenylyl cyclase

cAMP binds to regulator units of protein kinase A (PKA) and causes regulatory and catalytic subunits to separate (catalytic becomes active)

Example of covalent modification for regulation of enzyme activity

Glycogen phosphorylase helps break down glycogen

Phosphorylation activates glycogen phosphorylase

Regulation of enzyme activity phosphorylation

Adrenaline binds to its receptor that activates a g protein that activates adenylyl cyclase which produces cAMP from ATP.

cAMP activates PKA which phosphorylates target proteins

Ubiquitin Mediated Protein Degradation

E1 activates ubiquitin

E2 carries activated ubiquitin

E3 recognizes specific protein and transfers E2 ubiquitin to the target protein

What is α1 antitrypsin?

A protease inhibitor that inhibits elastase to protect lung tissue from excessive proteolytic activity (cigarette smoke oxidizes a methionine residue in α1 antitrypsin

what are carbohydrates composed of?

Carbon, hydrogen, and oxygen

What are the building blocks of carbohydrates and what do they form?

Monosaccharides are the building blocks

They can be linked to form disaccharides, oligosaccharides (3-10), and polysaccharides (over 10)

How are glycocojugates formed?

Glycoconjugates are formed through the covalent bond between carbohydrates and proteins / lipids

What is the role of carbohydrates?

They serve as major sources of metabolic energy for cells

Provide energy storage in the form of glycogen in animals and starch in plants

Provide carbon skeleton (biosynthesis of AA, nucleotides and more)

Function as recognition signals when attached to proteins and lipids (example blood types)

What is the major carbohydrate product produced by plants

Cellulose

What do carbohydrates contain structurally?

Multiple hydroxyl groups and may contain an aldehyde or ketone function group

Name this D-aldose

D-Glyceraldehyde

Name this D-aldose

D-Erythrose

Name this D-aldose

D-Threose

Name this D-aldose

D-Ribose

Name this D-aldose

D-Arabinose

Name this D-aldose

D-Xylose

Name this D-aldose

D-Lyxose

Name this D-aldose

D-Allose

Name this D-aldose

D-Altrose

Name this D-aldose

D-Glucose

L-Glucose

Name this D-aldose

D-Mannose

Name this D-aldose

D-Gulose

Name this D-aldose

D-Idose

Name this D-aldose

D-Galactose

Name this D-aldose

D-Talose

Name this D-Ketose

Dihydroxyacetone

Name this D-Ketose

D-Erythrulose

Name this D-Ketose

D-Ribulose

Name this D-Ketose

D-Xyulose

Name this D-Ketose

D-Psicose

Name this D-Ketose

D-Fructose

Name this D-Ketose

D-Sorbose

Name this D-Ketose

D-Tagatose

Most naturally occurring sugars?

D-sugars

Carbohydrates can occur in what forms?

Both linear and cyclic

Enantiomers

Mirror image stereoisomers (D and L)

D-glucose is known as?

Dextrose or blood sugar

Major fuel molecule used by cells to obtain energy

Mutarotation

The interconversion of α and β anomers through the open chain form in solutions

Furanose

5 membered ring

Pyranose

6 membered ring

Can adopt boat (steric interactions) or chair (best stability) conformations

Cyclization of monosaccharides occurs when?

A hydroxyl group reacts with the sugar’s carbonyl group

Aldoses react with alcohols to form?

Hemiacetals

Ketones react with alcohols to form?

Hemiketals

What are the glucose anomers?

α-D-glucose (down OH group) and β-D-glucose (up OH group)