Biochem Lecture 11: Enzymes as catalysts

Regeneration of the Enzyme Catalyst Last steps that correspond to this

•Again, the transition state is stabilized, making it form easily •Once formed, the transition state breaks down to the most stable products: a second peptide fragment and a free serine residue •Note the enzyme returns to its original form: a true catalyst Step 7: Second oxyanion tetrahedral intermediate Step 8: Acid catalysis breaks the acyl-enzyme covalent bond Step 9: the product is free to dissociate

J. Using a diagram of the reaction mechanisms of the serine protease class of enzymes and HIV protease enzyme, interpret the occurrences of: 1.substrate specificity 2.the proximity effect 3.the transition state 4.binding energy

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Enzyme forms weak interactions with substrate, liberating energy that is used to decrease the activation energy...but how?

Any time a weak interaction is formed (H-bond, ionic interaction, hydrophobic interaction, van der Waals), a tiny bit of energy is released. The enzyme will capture the sum of all these small energy changes and use this energy to lower the energy required to reach the transition state.

You are treating a patient who has a mutation in her hexokinase enzyme. Instead of a glutamate residue at position 256, she has threonine residue. Which of the following would be a likely effect on glucose binding to this altered enzyme? A.Glucose won't bind at all to the enzyme. B.Glucose will bind, but with a lower affinity than normal. C.Glucose will bind, but with a higher affinity than normal. D.Glucose will act as an irreversible inhibitor.

Glucose will bind, but with a lower affinity than normal. Recall that glutamate has an acidic side chain and is negatively charged. This can be seen in the given figure. Recall that threonine has a non-polar side chain. The structure isn't needed to answer the question. The polar and negatively charged side chain of glutamate makes key weak interactions with the glucose allowing it to bind with specificity and high affinity. Replacing those negative charges with a neutral side chain will decrease both the specificity and affinity. It will not prevent glucose from binding - there are still the many other amino acid side chains present that make key weak interactions. Certainly glucose won't bind with higher affinity, and as such it could never be an irreversible inhibitor.

Catalysis usually involves the action of one or more amino acid residues with reactive side chains that are specifically located near the substrate binding site. Which amino acids are usually involved?

Ser, Cys, His, Lys, Glu, Asp Sometimes cofactor molecules are required because they can drive more reactions than most amino acid side chains

C. Relate the structure of an enzyme active site of an enzyme to its function in terms of high selectivity for substrate and high specificity for the reaction it catalyzes.

Substrates bind at the active or catalytic site of the enzyme with the help of biomolecular recognition. When the substrate binds, weak interactions form. The enzyme will change its shape to accommodate the substrate. The structure of the enzyme active site matches the transition state of the substrate more and therefore will make more weak interactions with it. This drives the reaction to the TS. Once at the TS, the reaction go forward to make products or go back to the original substrate. Product formation is usually more energetically favorable and therefore more likely. Enzymes exhibit high specificity and selectivity because they often react with only a single molecule and even its stereoisomers are ignored. They react with a stereospecific substrate to give stereo specific products. Non specific chemical catalysts usually cause loss of stereochemistry. Catalytic activity depends upon the tertiary structure of the enzyme: one enzyme one function. Substrates with small changes will still bind, but they will not have the same high affinity.

Slide 16--explain the "stickase" example

The key here is understanding the interaction between the enzyme and the substrate (the stick). The goal is to break the stick into two pieces. This certainly won't happen spontaneously. As shown in part C, the substrate binds to the enzyme active site. There are a few weak interactions that allow the stick to bind. However, many more weak interactions can form if the stick bends. So, the favored conformation will be for the stick to bend and form all these weak interactions. But, in the process of bending, the stick reaches its transition state and readily breaks. The enzyme recognizes the stick is broken and releases the two pieces (products). Note that this works because the stick is drawn toward its transition state, which forms the maximum number of weak interactions between enzyme and substrate. In part B, if the substrate was a match to the enzyme active site, it would form maximum interactions for the ES complex, and there would be no driving force to form the transition state.

D. Relate, compare and contrast how the following affect the rate of an enzyme reaction: 1.enzyme concentration 2.substrate concentration 3.temperature 4.pH

1. More enzymes is faster, but needs substrate to bind to. one enzyme per substrate. 2. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity is reached. The leveling off reflects saturation of all available binding sites. 3. Reaction velocity will increase with temperature until a peak velocity is reached (more molecules have inc. energy to pass over barrier). Further elevation of temperature causes a decrease in reaction velocity due to denaturation of enzyme 4. pH effects ionization states of the active site. Extremes of pH can also lead to denaturation of the enzyme. The pH at which maximal enzyme activity is achieved is different for each enzyme and reflects the [H+] at which the enzyme functions

A patient has a defect in her chymotrypsin enzyme. In place of the catalytic triad histidine residue, she has phenylalanine residue. How will this difference affect the function of her chymotrypsin enzyme? A.No change - Phe is very similar in structure to His. B.Increase in Function - Phe is more hydrophobic and will be more effective at cleaving hydrophobic polypetides. C.Decrease in Function - Phe is only a weak base and cannot readily accept a proton from the nearby Ser residue needed to activate the Ser. D.Loss of Function - Phe is not a basic residue and cannot accept a proton from the nearby Ser residue needed to activate the Ser.

D.Loss of Function - Phe is not a basic residue and cannot accept a proton from the nearby Ser residue needed to activate the Ser. Notice that the histidine side chain is ionizable but the phenylalanine side chain is not. So Phe can't have the same function as His, so the catalytic triad won't work properly. Loss of Function

B. Relate enzyme deficiency to human disease.

Enzyme deficiency is the cause of many disorders. An enzyme with abnormal amino acid substitutions (that can change the structure or shape) may have abnormal activity and cause disease (PRPP synthetase and gout) An enzyme in a multi-enzyme pathway may be missing, causing the whole pathway to fail (OTC deficiency and the urea cycle).

A. Relate enzymes as catalysts and why they are important for most biological reactions to occur.

Enzymes are proteins that act as catalysts to speed up the rate of biochemical reactions. They do not change delta G or concentrations. Instead, they decrease the activation energy to make the reaction proceed faster. Most biochemical rxns will not proceed at any rate in the absence of an enzyme. Enzymes-catalyzed rxns proceed with very few or no side reactions. Chemical catalysts usually catalyze a number of unwanted side reactions. So enzyme catalysts are efficient. Most enzyme-catalyzed rxns are fully reversible too (like carbonic anhydrase)

Aspartyl Protease

Notice similarities and differences to chymotrypsin mechanism: activation of water for nucleophilic attack, no enzyme-bound intermediates, rearrangement of tetrahedral carbon intermediate leads to cleavage of peptide bond.

Lactate dehydrogenase removes 2 electrons and one proton from the molecule lactate to form the molecule pyruvate. What type of enzyme is lactate deyhdrogenase? A.Hydrolase B.Isomerase C.Ligase D. Oxidoreducatase

Oxidoreductase Notice - in this example lactate is oxidized to pyruvate (2 electrons are removed). If the reverse reaction is carried out - 2 electrons are given to pyruvate to reduce it to lactate - this is still an oxidoreductase reaction. Compare this to a ligase reaction, where two different molecule are connected together into one new molecule. If the reaction runs backwards, the one molecule is split into two pieces. That would be a lyase reaction.

E. Identify the six classes of enzymes and identify the reactions they catalyze. OTHLIL Oxen that have large igloos love enzymes!

Oxidoreductases -add or remove one or more electrons from or to substrates. Oxidoreductases are frequently called "dehydrongenases" because their oxidation reactions often remove two electrons and a proton from the oxidized substrate Transferases -transfer a group from one substrate to another. includes kinases that transfer phosphate groups from ATP to substrates. Hydrolases - carry out the hydrolysis of substrates. includes phosphatases that remove phosphate groups from substrates. Lyases - carry out the cleavage of a molecule into parts Isomerases - rearrange a substrate into an isomeric form (i.e., D to L form) Ligases - join molecules together by condensation, usually eliminating water

I. Relate the terms "binding energy," "proximity effect" and "induced fit" and relate these terms to an enzyme's ability to lower the activation energy of a reaction.

•Binding Energy: interaction of S with enzyme active site provides binding energy, bringing S closer in structure to the transition state. This lowers the activation energy and increases the reaction rate. •Proximity: Binding substrates increases their effective concentration within the active site, thereby increasing the reaction rate. •Induced Fit: Binding of S induces changes in the tertiary structure of E (conformational changes) that in turn distort S toward the transition state. •Orientation: geometry of S binding the active site will allow the reacting molecules to be in the perfect orientation for reaction, thus increasing the reaction rate.

Protease specificity

•Different protease enzymes cleave proteins at specific amino acids - specificity is determined by a substrate-binding pocket •Chymotrypsin: a hydrophobic binding pocket that favors aromatic residues and large non-polar residues (like Phe) •Trypsin: a deep binding pocket with a negatively charged residue (glu) at the bottom that favors the binding of lys and arg •Elastase has a very small binding pocket that favors binding of ala or gly residues (small and nonpolar amino acids) (AGE)

H. Recall an example of how pharmaceuticals can alter an enzyme-catalyzed reaction.

•Pharmacology: many drugs target the active site of enzymes. •Drug reacts with and destroys specific side chains in the enzyme active site. (Aspirin and COX 1) •OR...inactive precursor drugs are designed to target specific enzymes that modify the precursor into the active form of the drug.

Transition state stabilization Next 2 steps that correspond to this

•Specific interactions between the enzyme and the transition state provide stabilization, making it easier to form •Once formed, the transition state breaks down to the more stable products •The target peptide bond is cleaved *Binding energy: the multiple weak interactions that form between E and S liberate energy, which the enzyme uses to decrease the activation energy. Also, not literally shown, is the induced fit: The enzyme is undergoing conformational changes that will bend and stretch the target peptide bond to make it look more like the transition state, which facilitates cleavage of the peptide bond on the target protein. Step 3: The oxyanion tetrahedral intermediate is stabilized by hydrogen bonds Step 4: Cleavage of the peptide bond

Principles of Enzyme Catalysis: Chymotrypsin First 2 steps that correspond to this.

•Target protein binds to active site due to affinity of pocket for tyr residue + specific interactions between enzyme and substrate •Target peptide bond is in proximity to chemically active residues in the active site •The "catalytic triad" of residues cooperate to make the Ser-OH a strong nucleophile, which attacks the peptide carbonyl bond •The enzyme is a direct participant in the chemical reaction *This is a great example of the proximity effect: the individual amino acids that make up the catalytic triad are far apart in the primary sequence, but are right next to each other in the tertiary fold. Thus they can interact to provide an activated oxygen atom on the serine. Plus, the specificity pocket that binds the Tyr residue positions the substrate peptide perfectly in the active site so that the target peptide bond is right beside the activated serine oxygen. Together, this facilitates a highly specific and rapid reaction. Step 1: Substrate binding Step 2: Histidine activates serine for nucleophilic attack

G. Interpret an energy coordinate diagram for an enzyme-catalyzed reaction.

•The energy changes during a reaction can be described usingterm-8 a reaction coordinate diagram, a plot of the change in energy as a function of the reaction progress •The rate of a reaction is dependent upon the magnitude of the activation energy, Ea •Big activation energy? Slow conversion. Small activation energy? Fast conversion. •The structure of the substrate at the maximum energy is called the transition state and is usually intermediate between the structures of S and P. *Enzymes increase the rate of a reaction by decreasing the reaction activation energy ΔGo' (standard free energy change) is not changed, and so Keq is not affected: ΔGo' = - RT ln Keq

Activated Water breaks the bond Next steps that correspond to this

•The first product (a peptide piece) is released, whereas a fragment remains bound to the enzyme •The enzyme-substrate bond is broken by hydrolysis •The enzyme interacts with water, making it a stronger nucleophile, which attacks the carbonyl carbon. *Activated water? The histidine nitrogen's pair of electrons will attract an H+ of a water molecule, making the water oxygen more negative. That makes it a better nucleophile that will more readily attach the delta positive carbonyl carbon. Again: proximity and orientation. Step 5: The covalent acyl--enzyme intermediate Step 6: Water attacks the carbonyl carbon

F. Relate the free energy change for a chemical reaction and predict whether a reaction is spontaneous or non-spontaneous based on this free energy change.

•The free energy change for a reaction determines the spontaneity of a reaction, i.e., which direction a reaction is likely to proceed •The change in Gibbs free energy (ΔGo') for a reaction is the difference in energy between the products and the substrates. •ΔG < 0, the reaction proceeds in the forward direction spontaneously •enzyme catalysis changes the rate at which the equilibrium in the reaction is achieved but does not change the position of the equilibrium. •The equilibrium position is determined by the free energies of substrates and products -enzymes do not affect free energy change!!!

serine proteases

•a family of enzymes that hydrolyze specific peptide bonds in proteins (i.e., splits the protein into smaller polypeptides) •Each enzyme in the family hydrolyzes amide bonds adjacent to specific amino acid residues. •Main area of function: Digestion •Serine proteases are synthesized in the pancreas as pro-enzymes or zymogens that are initially inactive. •These enzymes are activated after they are released from the pancreas.