Lecture 4: Enzymes and Catalytic Mechanisms (BioChem)

Types of Reversible Inhibition

1. Competitive: Substance that resembles the normal substrate competes with the substrate for the active site. - Km: increases (less substrate affinity) - Vmax: unchanged 2. Noncompetitive: binds only to enzyme-substrate complex, locking the substrate in the enzyme preventing its release - Km: decreases (More substrate affinity) - Vmax: decreases (s cannot out compete i) 3. Uncompetitive (Mixed): The inhibitor binds to both the enzyme and the ES complex (50/50) - Km: Unchanged - Vmax: Reduced

3 Mechanisms of Enzymatic Catalysis

1. Covalent Catalysis 2. Acid-Base Catalysis 3. Metal Ion Catalysis

7 Steps of the Chymotrypsin Mechanism.

1. Substrate binding and generation of a serine nucleophile 2. Nucleophilic attack and formation of tetrahedral intermediate 3. Formation of Acyl Enzyme Intermediate 4. First product Departs 5. Water Enters the Active Site 6. Create of a new nucleophile (from water) and nucleophilic attack 7. Break off and Product Dissociation

Assumptions of Machaelis-Menton Equation

1. We use Vo to compare velocities of reactions - Also only moving forward in reaction, making products 2. Only works for enzymes with single substrate binding (only one binding site) 3. Steady state Assumption. - It doesn't directly go to product it goes through the enzyme substrate complex, so [ES] is in equilibrium with both 3. Vmax occurs when the enzyme is saturated by substrate 4. Vmax varies with the amount of enzyme that is used 5. Km is a measure of an enzymes affinity for its substrate 6. Km is inversely related to affinity

1. Why would an enzyme want to be perfect? 2. Why aren't all enzymes perfect?

1. Why would an enzyme want to be perfect? - If a product of enzymatic reaction needs to be made very quickly - To get rid of something that when builds up is toxic 2. Why aren't all enzymes perfect? - Not enough selective pressure (evolutionarily) to make them all perfect

Chymotrypsin Active Site

3 amino acids that form a catalytic triad 1. Aspartate 2. Histidine (ring, basic, pH 6 loses proton, neutral) 3. Serine (hydroxyl, polar) (protein folding lets them interact with eachother, they are not right next to each other in primary sequence)

perfect enzyme

A perfect enzyme is only limited by the rate of diffusion of a substrate into the active site of the enzyme. - The rate of the reaction is so fast that the rate-limiting step is the diffusion instead The diffusion limit represents an absolute constraint on evolution These max out Kcat / Km - Where the ratio is close to the diffusion controlled rate - Around 10^8

1. Covalent Catalysis

A transient covalent bond is formed between the enzyme and the substrate - This Changes the reaction pathway The covalent complexes always undergo further reaction to regenerate the free enzyme Photo: We have A-B attached and want to break them apart 1. B can act as a leaving group, with Enzyme attaching to A (bond broken) 2. Water comes in breaking bond between Enzyme and A, releasing A

Active Site / Substrate

Active Site: Where enzyme-catalyzed reactions occur - very protective site, a protected environment for reactions to occur Substrate: the molecule that is bound in the active site and acted upon by the enzyme - Very specific for only one kind of substrate. - Even specific for the L vs. R

Kinetic Properties of Allosteric Enzymes

Allosteric proteins: having "other shapes" or conformations Allosteric Enzymes: function through reversible, noncovalent binding of regulatory compounds called allosteric modulators (or effectors) - modulators may be inhibitory or stimulatory - Conformational changes induced by one or more modulators interconvert more active and less active forms of the enzyme Homotropic: If substrate is the modulator (substrate causes the conformational change) Heterotropic: the modulator is a molecule other than the substrate Different subunits have different active sites (that can be cooperative) Allosteric enzymes are typically larger and more complex than nonallosteric enzymes with two or more subunits - they do not obey Michaelus Menton Kinetics

Aspartate Transcarbamoylase

An allosteric Enzyme This enzyme adds aspartate to Carbamoyl Phosphate to produce carbamoyl aspartate. - Eventually (many steps down) produces nucleotides - first step that produce CTP Has an active T-state and inactive R-state Example of Negative Feedback - If CTP present it regulates and binds to enzyme produces inactive conformation - CTP low it will be in active state

Enzymes

Biological Catalysts that increase reaction rates without being used up. Almost all are globular proteins - Although some RNA also catalyze reactions

Biological vs. Chemical Catalysts

Biological Catalysts works better: 1. Greater reaction specificity: avoids side products - specific for reactants and products produces - chemical often gives you a racemic mixture 2. Milder reaction conditions: conducive to conditions in cells - effective under mild reactive conditions - chemical catalysts often require a lot of heat or low pH's etc. 3. High reaction rates: in a biologically useful time - Happens very quickly 4. Capacity for regulation: control of biological pathways - can add one phosphate group for example to regulate its activity - Enzymes can change shape/ structure to change function

How do enzymes lower activation energy?

By having many favorable interactions between the enzyme and the substrate to overcome the energy of activation. 1. Covalent Interactions - Functional groups on enzyme form transient covalent bonds with a substrate to activate it for reaction (or a functional group may be temporarily transferred) - Covalent interactions b/w enzymes and substrates lower the Ea by providing an alternative, lower energy path 2. Non-covalent Interactions - Mediated by the same forces that stabilize protein structure (H bonds, hydrophobic interactions) - Weak interactions can be critical for initial binding of enzyme to substrate - Weak interactions are more important though during the transition state. - Formation of each weak interaction is accompanied by release of a small amount of free energy that stabilizes the reaction - Most binding energy comes from these interactions

Activation of Chymotrypsin

Chymotrypsin is synthesized as a single polypeptide chymotrypsinogen which is inactive (inability to fold) Once released from the pancreas, it is activated by the by the cleavage of the polypeptide to yield 3 chains, which with the help of sulfide bridges, can fold into its active form. What is the purpose of making this inactive "zymogen" form first? - Proteases digests proteins, so you only want it to be activated in the small intestine, not in the pancreas.

Chymotrypsin

During digestion, dietary products must be broken down into small peptides by proteases. - Proteases assist in breaking down peptide bonds. Chymotrypsin is one of the main pancreatic proteases; it cuts peptides at a specific location, adjacent to large aromatic amino acids. Polypeptides are more high energy than single amino acids, So this reaction is spontaneous in water without chymotrypsin, but it will take 100,000 years. So enzymes increase the rate of the reaction.

Would Chymotrypsin work in the stomach?

Enzymes have an optimum pH (or pH range) at which their activity is maximal Amino acid side chains in the active site may act as weak acids and bases with critical functions that depend on maintaining a certain ionization (depending on pH) At a different pH the histidine will be at a different pH that will change is protonation, as well as aspartate.

How do Enzymes Work?

Enzymes increase the rate of reaction by lowering the activation energy barrier and allow molecules of relatively low energy to take part in reactions at body temperature Does not change the spontaneity of reaction (G)

Mixed Inhibition

Inhibitor binds to some fraction of the Enzyme Substrate complex or the enzyme itself - neither of which is at the active site. Vmax is lowered (cannot outcompete I with S) Km depends on the relative affinity of the mixed inhibitor for the E vs. ES - can lead to increase, decrease or no change in Km

2. Acid Base Catalysis

Involves the movement of protons inside of an active site. Active sites may contain residues that can participate in hydrogen ion transfer. By transferring a hydrogen ion, an active site may: 1. Activate nucleophiles required for catalysis 2. Stabilize charged groups 3. Increase electric interactions that may stabilize the transition state

Enzyme Inhibition

Irreversible Inhibitors (inactivators) react with the enzyme: - One inhibitor molecule can permanently shut off one enzyme molecule - They are often powerful toxins but also maybe used as drugs (nerve gas, penicillin) Reversible Inhibitors bind to and can dissociate from the enzyme: - They are often structural analogs of substrates or products - They are often used as drugs to slow down a specific enzyme

Catalytic Effiency

Kcat/Km [unit 1/(s * M)] - A useful metric to compare efficiencies of different enzymes Large ratio of Kcat to km - Km affinity of enzyme of substrate (lower, more affinity) - Kcat: turnover of enzyme Best possible enzyme: super high affinity (low km) and super fast turnover (super high kcat)

Kinetics of Competitive Inhibition

Km Increases: - affinity of substrate, goes down as you add inhibitor - takes longer to get to 50% enzyme-substrate Vmax does not change: - When [s] far exceeds [I] the probability that the inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax. - You can outcompete the inhibitor eventually with more [s] Lineweaver Burk Plot: - Y intercept (1/Vmax): stays the same - X intercept (-1/km): Number gets smaller - Slope (km/vmax): increases gets more steeper

Uncompetitive Inhibition Kinetics

Km: Reduced - Affinity for the substrate goes up. The inhibitor locks the substrate in. Vmax: Reduced - You can't outcompete inhibitor with substrate, once its attached there is nothing you can do. Lineweaver Burk Plot: - Y intercept (1/Vmax): Increases - X intercept (-1/km): Increases - Slope (km/vmax): Same

Noncompetitive Inhibition (Mixed) Kinetics

Km: will not change - equation is pulled equally on both sides creates same shift (le chattlier) Vmax: Decrease - cannot outcompete inhibitor with substrate Lineweaver Burk Plot: - Y intercept (1/Vmax): Increase - X intercept (-1/km): Does not change - Slope (km/vmax): Increase

3. Metal Ion Catalysis

Less Important (know bolded!) Metal ions such as zinc, magnesium, and iron are used as cofactors by a multitude of different enzymes The positive charge on metal atoms allow them to: 1. Assist in forming strong nucleophilic species 2. Stabilize structures during the reaction - by forming coordinate bonds 3. Hold the substrates inside the active site.

Lock and Key Model

Lock and Key: Enzymes are specific. The only wok on the substrate that they "fit perfectly" Just like a lock has a specific key to open it. - What are the limitations? doesn't account for conformational changes we see. Induced Fit Model: Substrate fits in pretty well, but after it binds there is a conformational change in the enzyme that makes it fit even better. - Initial binding is good but not perfect, best binding due to conformational change

Why are vitamins important?

Most Coenzymes are derived from vitamins As chemical partners for the enzymes involved in the body's metabolism, cell production, tissue repair, and other vital processes (metabolic processes)

2. Chymotrypsin Mechanism: Nucleophilic Attack

Nucleophilic attack and formation of tetrahedral intermediate Alkoxide Ion (O-) on serine now nucleophilic attacks the carbonyl on the peptide bond (electrophile). Covalent bond forming between the Oxygen of the Serine with our substrate (peptide bond) Transition State: double bond breaks on carbonyl forming negatively charged oxygen. Oxyanion Hole: Stabilizes the transition state - Has partially positively charged Nitrogen in it (from amine groups) - positively charged nitrogens interact favorably with the negatively charged Oxygen, stabilizing the transition state

cofactors and coenzymes

Some enzymes require an additional chemical component (one or both) for activity 1. Cofactor: one or more inorganic ions (Fe2+, Mg2+) 2. Coenzyme: complex organic molecules - Generally larger than Cofactors - Often act as transient carriers of specific functional groups - Prosthetic group: a coenzyme that is tightly (or even covalently) bound to the enzyme Holoenzyme: A complete catalytically active enzyme together with its bound coenzyme and/or metal ions Apoenzyme: The protein part of an active enzyme together with bound coenzyme and/or metal ion.

Competitive Inhibition

Substance that resembles the normal substrate competes with the substrate for the active site. - Many are structurally similar to the substrate and combine with the enzyme to form an ( El)complex. (without leading to catalysis) When [s] far exceeds [I] the probability that the inhibitor molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax

1. Chymotrypsin Mechanism: Substrate Binding

Substrate binding and generation of a serine nucleophile Turn Serine into a really good nucleophile. (must remove proton) - OH is a bad nucleophile - but O- is a great nucleophile When substrate binds there is a conformational change that brings aspartate 2a closer to histidine (Induced fit) - Hydrogen bond forms between partially +H on histidine and partially - oxygen of the carbonyl of Aspartates R group This changes the pKA of histidine from 6 to 11 (now positively charged) - This rapid change causes histidine to become protonated by grabbing the H off of serine - Turning Serine into a nucleophile Alkoxide Ion (hydroxyl group loses its proton. OH to O-)

3. Chymotrypsin Mechanism: Formation of Acyl Enzyme Intermediate

Tetrahedral Intermediate is very unstable, so even when stabilized by the oxyanion hole, it will not last very long. The electrons from the Carbon-Nitrogen bond act as a nucleophile and grab the Hydrogen off the histidine, breaking the bond. Peptide Bond Breaks!

4. Chymotrypsin Mechanism: First product Departs

The first produce the amine group leaves

Uncompetitive (Type of Mixed) Inhibition

The inhibitor binds to both the enzyme and the ES complex (50/50) - Never binding at the active site.

Velocity of a Reaction

The rate V is: - the quantity of A that disappears / time - The quantity of product made / time V = k [S] [S] = substrate concentration k = rate constant (units 1/s) k is inversely related to the activation energy k = Ae^-(Ea/RT) k and velocity of reaction are inversely proportional to Ea, - Ea up, k down, v down - Ea down, k up, v up

Kinetics

The study of the rates of chemical reactions

6. Chymotrypsin Mechanism: Nucleophilic attack by Water

This nucleophile (OH-) created from the water molecule then attacks the carbonyl on the Acyl-enzyme This breaks the double bond leaving an negative charge on Oxygen, forming a tetrahedral intermediate. This intermediate is stabilized again by an oxyanion hole. - Positive nitrogens stabilize negative Oxygen

Uncompetitive Inhibition

Uncompetitive Inhibitor binds only to enzyme-substrate complex - It does not bind at the active site - It locks substrate in enzyme preventing its release (increasing affinity b/w enzyme and substrate ) Creates Enzyme, substrate, inhibition complex

Apoenzyme

protein portion of an enzyme Apoenzyme: The protein part of an active enzyme together with bound coenzyme and/or metal ion.

Lineweaver Burk Plot

the double reciprocal graph of the Michaelis-Menten equation Equation of line X axis = 1/ [s] y axis = 1/Vo Y intercept = 1/Vmax X intercept = -1/km Slope = km / Vmax Only holds true for enzymes that obey michaelis menton kinetics

Enzyme Kinetics

the study of the rates of enzyme-catalyzed reactions

Binding Energy

∆Gb: The energy derived from enzyme-substrate interactions (both covalent and weak) - Binding energy is a major source of free energy used by enzymes to lower the Ea's of reactions - most comes from weak interactions Works in 2 to lower the Ea: 1. Increases Substrate Proximity - substrates proximity with other substrates, want them to be close to each other to react quickly. - Can result from both covalent and non-covalent interactions 2. Stabilize the Transition State - Transition state is the least stable point of reaction and limits the rate - Lower Ea by stabilizing this state through weak interactions. - Weak interactions are critical during the transition sate - This model is also compatible with the "induced fit" model of substrate binding.

kCAT

kCAT = Vmax / [E] turnover number (molecules catalyzed per second in optimal conditions) - Units: (1/s) Vmax is not an inherent property of an enzyme - It is dependent on the amount of enzyme present. kCat is a physical property inherent to an enzyme - Each enzyme has a unique kCAT

Most Important Parts of Reaction to Know: 1. Induced Fit 2. Acid/base Catalysis 3. Covalent Catalysis 4. Transition state stabilization 5. Substrate specificity

Understanding how chymotrypsin assists in breaking peptide bonds 1. Induced Fit - Step 1: substrate binding causes aspartate to get closer to histidine, conformational change happened changing position of amino acids in active site. 2. Acid-Base Catalysis (protons move in active site) - Step 1 (Base Catalysis): Histidine grabs proton from Serine - Step 3: Tetrahedral Intermediate grabs H off of histidine forming Amide. - Step 5 (Base catalysis): Histidine steals H from Water - Step 7: Tetrahedral Intermediate grabs H off of histidine forming carboxylic acid product 3. Covalent Catalysis (Must be between enzyme and substrate) - Step 2: covalent bond formed b/w Serine (O-) and carbonyl on peptide bond (nucleophilic attack) - Not step 6 b/c that is between water and substrate not enzyme/substrate 4. Transition State Stabilization - Step 2: Oxyanion Hole, partially positively charged nitrogen stabilizes negative oxygen. - Step 6: Second transition state also stabilized by oxyanion hole. 5. Substrate Specificity - Chymotrypsin only cleaves peptide bond next to aromatic amino acids. - This happens because the aromatic group fits perfectly into the S1 pocket (big and hydrophobic), this pocket sits next to the active site and allows for substrate specificity by regulating.

Velocity of Reaction

V = k [s]

Machaelis-Menton Equation

Vo = Vmax * [S] / Km + [S] Km: substrate concentration at which your enzyme is operating at 50% is maximum velocity (1/2 Vmax) - Inverse of affinity of Enzyme for substrate Vmax: velcocity when enzyme is completely saturated with substrate. - If you add more substrate you won't increase Velocity

5. Chymotrypsin Mechanism: Water enters the active site

Water molecule moves in and positions itself in the same spot that the amide was in. Histidine grabs the H off of the water molecule, creating a hydroxide ion (OH-) which is a strong nucleophile

[S] over time

We like to think of [S] concentration as constant, but actually overtime as the reaction progresses, the amount of substrate changes. - Linear relationship at beginning of reaction with product produced and time, but this changes When we compare velocities we often use Vo (V not, V initial)

Holoenzyme

apoenzyme + cofactor Holoenzyme: A complete catalytically active enzyme together with its bound coenzyme and/or metal ions