Ch. 6 - Enzymes (catalysis)

Eduard Buchner first discovered enzymes in what year?

1897

What does the word "enzyme" mean in Greek?

"in yeast"

Why doesn't diamond suddenly convert to graphite even though it's thermodynamically favored?

Because of the high activation energy barrier required for the conversion

What is the difference between kinetics and thermodynamics in regards to reactions?

Thermodynamics determines if a reaction will occur, kinetics determines how fast it occurs

What determines if a reaction is spontaneous?

The ΔG (Gibbs free energy change)

What determines how fast a reaction occurs?

The activation energy (Ea)

What is activation energy (Ea)?

The energy needed to reach and overcome a high energy transition state in a reaction

With lower activation energy, what happens to the reaction rate?

More molecules can achieve that energy, resulting in a faster reaction rate

What is a catalyst?

A chemical that speeds up the rate of a reaction without itself being consumed

How do enzymes increase the rate of biochemical reactions?

By lowering the free energy of the transition state and thus lowering the activation energy (Ea)

Alternatively, how else can enzymes lower activation energy?

By providing an alternate route with a lower energy transition state

For a reaction with 2 transition states, which one does the enzyme need to catalyze?

The enzyme must lower the free energy of the highest energy transition state

What are the 2 components to a chemical reaction?

Kinetic (rate) component and thermodynamic (energy) component

Which component are enzymes primarily concerned with?

The rate component (kinetics)

What are the main things enzymes DO?

1) Lower energy of activation 2) Speed up time to reach equilibrium 3) Stabilize the transition state

What should enzymes NOT change about a reaction?

The ΔG (Gibbs free energy/energy yield) of a reaction

What should enzymes NOT change about a reaction?

The equilibrium constant for the reaction

By what factor can enzymes accelerate biochemical reactions compared to non-enzyme catalysts?

10^9 to 10^20

Under what conditions can enzymes catalyze reactions?

Mild conditions like pH 7.4 and 37°C

What advantage do enzymes have regarding side products?

They conduct reactions with no side products

How can the activity of enzymes be controlled?

Their catalytic activity can be regulated

What is the lock and key model of enzyme substrate binding?

The enzyme is the lock and the substrate is the key - explains enzyme specificity

Who proposed the lock and key model?

Emil Fischer in 1899

What is the main limitation of the lock and key model?

It doesn't account for enzymes that bind diverse substrates and doesn't explain transition state stabilization

What is the induced fit model of enzyme substrate binding?

When substrate binds, it induces a conformational change in the active site that stabilizes the transition state

Who proposed the induced fit model?

Koshland in 1958

What does the induced fit model explain that lock and key doesn't?

Why more than one substrate can be recognized by certain enzymes - because the enzyme can fit around different substrates

What is a cofactor?

An additional chemical component (non-protein) required for some enzymes to function

What are the types of cofactors?

Metal ions or small organic molecules called coenzymes

What do coenzymes often come from?

Vitamins (organic nutrients required in small amounts in diet)

What is an apoenzyme?

The protein component of an enzyme

What is a holoenzyme?

The apoenzyme plus the cofactor combined

What is a prosthetic group?

A tightly bound cofactor to an enzyme

What are the main factors affecting enzyme activity?

1) Temperature 2) pH 3) Presence of cofactors 4) Allosteric effectors 5) Poisons/inhibitors

What are the 6 mechanisms of enzymatic catalysis?

1) Proximity effects 2) General acid/base catalysis 3) Covalent catalysis 4) Electrostatic stabilization 5) Preferential TS stabilization 6) Metal ion catalysis

What does proximity effects do for enzyme catalysis?

Positions molecules close together and orients them for reaction, freezing out rotational and translational motions

What is general acid/base catalysis (GABC)?

Donating/accepting a proton via weak acid/base groups to stabilize unstable TS and lower Ea

Which amino acid residues can participate in GABC?

Those with pKa values near physiological pH - Glu, Asp, Lys, Arg, Cys, His, Tyr

What is covalent catalysis?

When enzyme active site provides a nucleophile that forms a covalent enzyme-substrate adduct, altering the reaction pathway

Which amino acids commonly form nucleophiles in covalent catalysis?

Serine, cysteine, lysine, and histidine

What is electrostatic stabilization?

Charge distributions in active site that electrostatically stabilize an unstable charged transition state

What is preferential stabilization of strained transition state?

Lowering Ea by stabilizing a conformationally unstable transition state

What is transition state strain?

Another name for preferential stabilization of strained transition state

What are the two types of metal ion catalysis?

Metalloenzymes (tightly bound metal ions) and metal-activated enzymes (loosely bound ions)

What metals are examples of metalloenzymes?

Fe++, Fe+++, Cu++, Zn++, Mn++, Co++

What ions are examples of metal-activated enzymes?

Na+, K+, Mg++, Ca++

What are the 4 ways metal ions aid in catalysis?

1) Charge shielding 2) Binding substrates to orient them 3) Through redox reactions 4) Ionizing water to make it better nucleophile

What is ribonuclease A and what does it do?

An enzyme that catalyzes RNA hydrolysis, exhibits TS stabilization and uses GABC

What amino acids are in the active site of ribonuclease A that perform GABC?

His 12 (general base) and His 119 (general acid)

What is lysozyme and what does it catalyze?

A small globular protein that destroys polysaccharide chains of bacterial cell walls by hydrolyzing glycosidic bonds

What three catalytic mechanisms does lysozyme employ?

1) GABC by Glu35 2) Transition state stabilization of residue D in distorted ½ chair conformation 3) Covalent catalysis by Asp 52

What nucleophilic groups on serine proteases perform covalent catalysis?

Serine residue in the active site

What is the catalytic triad of serine proteases?

Three residues working together: Asp, His, and Ser

Which serine proteases have specificity?

Trypsin (deep negative charge), chymotrypsin (deep hydrophobic), elastase (shallow uncharged)

What is TPCK and what does it do?

Tosyl-L-phenylalanine chloromethyl ketone - a reactive substrate analog that proves His 57 is important in chymotrypsin

What is TLCK?

Tosyl-L-lysine chloromethyl ketone - reacts with trypsin to prove His 57 is important

What is DIPF and what does it do?

Diisopropylphosphofluoridate (nerve gas) - irreversibly inhibits serine proteases by reacting with Ser 195

What is the general name for enzymes with Ser in the active site performing covalent catalysis?

Serine protease family

What is an oxyanion hole?

A structure that electrostatically stabilizes the oxyanion intermediate in serine protease catalysis

What group provides the opposite charge in the oxyanion hole?

Backbone amide NH's provide δ+ charge to stabilize O- charge

What is a zymogen?

An inactive enzyme precursor with extra protein blocking the active site

What converts zymogens to active enzymes?

Proteolysis by a protease

What are zymogens also called?

Proenzymes

Why are proteases often synthesized as zymogens?

To prevent self-digestion in the pancreas

What is trypsinogen and what activates it?

Inactive trypsin precursor activated by enteropeptidase or trypsin autoactivation

Which enzyme activates many pancreatic proteases?

Trypsin

What is the pancreatic trypsin inhibitor and what does it do?

A protein that binds trypsin very tightly to prevent trypsin from digesting the pancreas

What is acute pancreatitis caused by?

Premature activation of pancreatic zymogens

What are the major classes in the IUBMB enzyme classification?

1) Oxidoreductases 2) Transferases 3) Hydrolases 4) Lyases 5) Isomerases 6) Ligases

What do oxidoreductases catalyze?

Oxidoreduction reactions (transfer of electrons)

What is the substrate that is oxidized called?

The hydrogen donor

What is NAD+ and what is it used for?

Nicotinamide Adenine Dinucleotide - cofactor for oxidoreductases

What is NADP+ and how does it differ from NAD+?

Nicotinamide Adenine Dinucleotide Phosphate - same as NAD+ but phosphorylated

What are FAD and FMN?

Flavin adenine dinucleotide and flavin mononucleotide - cofactors for redox reactions

What do transferases do?

Transfer functional groups from donor molecules to acceptor molecules

What is an example of a transferase reaction?

Hexokinase transferring phosphate from ATP to glucose

What do hydrolases do?

Cleave bonds by adding water

What is an example of a hydrolase?

Proteases like trypsin and chymotrypsin

What is carboxypeptidase A and what does it do?

A hydrolase that removes amino acids from the carboxyl terminus of proteins

What do lyases do?

Add water, ammonia, or carbon dioxide across double bonds, or remove these to produce double bonds

What is aldolase and what does it do?

A lyase that breaks C-C bonds in fructose-1,6-bisphosphate

What is aconitase and what does it catalyze?

A lyase that converts citrate to cis-aconitate to isocitrate

What is important to remember about lyase products?

The product must have a double bond

What do isomerases do?

Change the arrangement of groups within molecules to yield isomeric forms

What is an example of an isomerase?

Phosphoglucose isomerase converts glucose 6-phosphate to fructose 6-phosphate

What do ligases do?

Tie molecules together by forming new C-C, C-S, C-O, and C-N bonds, requiring energy from ATP

What is pyruvate carboxylase and what does it catalyze?

A ligase that converts pyruvate to oxaloacetate using ATP and HCO3-

What is the key to identifying ligases?

Look for energy from ATP or NTP cleavage

Why is it important not to confuse synthetases and synthases?

Synthetases are ligases (need ATP), synthases are lyases (don't need ATP)

What is a transition state analog (TSA)?

A stable analog that resembles the transition state conformation of a reaction

How do transition state analogs bind to enzymes?

Very tightly, causing enzyme inhibition

What do transition state analogs provide evidence for?

That enzymes preferentially bind and stabilize transition states

What is deoxycoformycin and what is it used for?

A TSA of adenosine deaminase - FDA approved for leukemia treatment

What is adenosine deaminase (ADA)?

An enzyme that deaminates adenosine to inosine

What is the mechanism of deoxycoformycin inhibition?

It binds much more tightly than the substrate because it resembles the tetrahedral transition state

What is carbonic anhydrase and what does it catalyze?

A metalloenzyme that catalyzes: H2O + CO2 ↔ H+ + HCO3-

How does carbonic anhydrase use a metal ion?

Zn2+ polarizes water to make it a better nucleophile

What is the EC number for carbonic anhydrase?

EC 4.2.1.1

What role does carbonic anhydrase play in the body?

Makes CO2 soluble in blood for transport

What is the blood buffering system?

The bicarbonate buffer system using carbonic anhydrase and the Henderson-Hasselbalch equation