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Enzymes
Biomed 2
| Question | Answer |
|---|---|
| How are enzymes able to speed up reactions? | Induced fit between correct substrate and enzyme |
| What is the implication of an induced fit between correct substrate and enzyme? | Electrostatic interactions to form between the two, strain to be placed on shape of substrate that helps better approximate shape of the TS, correct positioning of catalytic groups in the enzyme |
| Describe the electrostatic interaction in the absence of water and why this occurs? | Stronger in absence of water - water tends to be displaced from active site when a substrate enters |
| What are two ways in which catalytic groups may speed up reactions? | Acid-base catalysis or covalent catalysis |
| What is the implication of using an acid/base as a catalyst? | Adds or removes a proton to make a substrate more reactive |
| What can act as an acid/base | Side chains of certain amino acids |
| What is an amino acid that can function easily as an acid or base? | Histidine |
| In ester hydrolysis, explain what happens when there is an absence of an enzyme. | Water would be used as a nucleophile which would knock the -RO group (forming -ROH) and attach itself to the carbon. However, because the O in water is a weak nucleophile, the reaction would need heat to occur. |
| In ester hydrolysis, explain what happens when there is an enzyme present | Hydroxide is a much better nucleophile so would be able to knock off the -RO group which would then attach itself to an H from water thereby regenerating hydroxide enzyme |
| In ester hydrolysis, explain what happens when the enzyme can act as a base | The base would remove the hydrogen from water, creating hydroxide (much more reactive). Hydroxide will then displace the -RO group. Base will then release hydrogen to -RO (creating -ROH), regenerating itself. |
| In ester hydrolysis, explain what happens when enzyme can act as an acid. | Acid donates H to carbonyl O (breaking double bond) so water can attack C and displace -RO group. H is then knocked off water and given to -RO. While H from carbonyl O is given back to acid. |
| Is it possible to have an enzyme act both an acid and base? | Yes |
| What is covalent catalysis? | When a nucleophilic side group in the enzyme active site forms a temporary covalent bond with the substrate |
| What are common nucleophilic side groups for covalent catalysis? | Asp/Glu and Ser/Cys |
| Ser and Cys are weakly nucleophilic so why are they a common nucleophilic group during a reaction? | Because their nucleophilicity is enhanced by presence of other AA's that can remove H |
| Though the other AA's are often distant from each other in the primary protein structure, how do they still have an effect on each during during covalent catalysis? | Because structure dictates function |
| How do distant AA's in primary protein structure have an effect on each other in chymotrypsin? | Enzyme breaks peptide bond s beside aromatic AA's → Final 3D structure creates a shape that will fit aromatic AA's which positions distant Asp, His, and Ser closer together. Electrostatic interaction allows for reaction |
| Describe how the electrostatic interactions between AA's cleave a peptide bond in chymomtrypsin | Negative charge on Asp pulls non H in His which then picks up an H from Ser - making Ser more nucleophilic. Ser can now form a temporary covalent bond with aromatic AA in substrate. Electrons shift, breaking peptide bond. |
| What happens after the peptide bond is broken during covalent catalysis of chymotrypsin? | Substrate picks H back up from His. Still need to release rest of substrate and regenerate enzyme so will use water. H from water goes to His, becoming more nucleophilic and bond with carbon. Electrons shift, Ser picks H back from His |
| What are cofactors typically? | Metal ions |
| What are four ways in which cofactors help enzymes speed up reactions? | Helps position substrate in active site of enzyme, polarize reactants to make them more likely to participate in rxn, can stabilize negative charges on substrate or the TS to make easier for nucleophilic attack, can accept/donate electrons in redox rxn |
| How does zinc help carbonic anhydrase act as a cofactor? | Substrate polarization and positioning - Zn binds to enzyme, polarizing water and correctly positioning resultant -OH (better nucleophile than water) |
| What is an example of a cofactor that stabilizes a charge of a substrate? | Magnesium |
| Describe how Magnesium acts as a cofactor in the formation of Glucose-6-Phosphate | Mg stabilizes negative charges on ATP, making more susceptible to nucleophilic attack and also helps speed up reaction by acting as a base |
| What is an example of a redox reaction that requires a cofactor | ETC |
| Describe how a cofactor helps ETC | Iron helps transfer electrons between enzyme and oxygen. |
| What has iron during ETC? | Cytochrome |
| What are coenzymes typically? | Derived from vitamins |
| How do coenzymes help a reaction? | Help carry out specific processes during a reaction |
| What are examples of specific proccesses that require coenzymes? | 1-C transfers, methylation, redox |
| What vitamin is responsible for 1-C transfers? | B9 |
| What vitamin is responsible for methylation? | B9/B12 |
| What vitamin is responsible for redox? | B3/B2 |
| What redox reaction requires B2? | FAD <-> FADH2; FMN <-> FMNH2 |
| What rexox reaction requires B3? | NAD+ <-> NADH + H+; NADP+ <-> NADPH + H+ |
| What is the basic difference between the types of rexo reactoins these forms as B3 help catalyze? | NADH - catabolic; NADPH - anabolic |
| What is the coenzyme form and reaction it promotes for vitamin B1 (thiamine)? | Thiamine pyrophosphate; decarboxylation, aldehyde group transfer |
| What is the coenzyme form and reaction it promotes for vitamin B2 (riboflavin)? | FAD, FMN; redox |
| What is the coenzyme form and reaction it promotes for vitamin B6 (pyridoxine)? | Pyridoxal phosphate; amino group transfer, transfulhydration, cleavage |
| What is the coenzyme form and reaction it promotes for vitamin B3 (nicotinic acid/niacin)? | NAD, NADP; redox |
| What is the coenzyme form and reaction it promotes for vitamin B5 (Pantothenic acid)? | Coenzyme A; acyl transfer |
| What is the coenzyme form and reaction it promotes for biotin? | Biocytin; carboxylation |
| What is the coenzyme form and reaction it promotes for vitamin B9 ( folic acid)? | Tetrahydrofolic acid; one-carbon group transfer |
| What is the coenzyme form and reaction it promotes for vitamin B12? | Deoxyadenosylcobalim, methylcobalamin; intramolecular rearrangements, hydrogen and methyl group arrangements |
| What is the coenzyme form and reaction it promotes for vitamin C (ascorbic acid)? | Unknown; hydroxylation |
| What is the coenzyme form and reaction it promotes for vitamin A? | Retinal; vision, growth, and reproduction |
| What is the coenzyme form and reaction it promotes for vitamin D? | 1, 24- dihydroxycholecalciferol; calcium and phosphate metabolism |
| What is the coenzyme form and reaction it promotes for vitamin E? | Unknown; lipid antioxidant |
| What is the coenzyme form and reaction it promotes for vitamin K? | Unknown; bloodclotting |
| Describe the role of cofactors and coenymes in the conversion of ethanol to acetaldehyde | Alcohol dehydrogenase is the enzyme. Cofactor (Zn) helps polarize and position substrate. Coenzyme (B3) helps with redox. |
| What is the optimal temperature for an enzyme? | Temperature of the organism |
| Do you think fevers are good or bad? | Good - helps kill off infection; Bad - deviated away from optimal temperature |
| What is the effect of pH for enzymes? | Changing pH can change protonation state of the enzyme and/or substrate |
| Pepsin is proteolytic at a low pH, where in the body would this be found? | Stomach |
| Chymotrypsin is proteolytic at a higher pH, where in the body would this be found? | Small intestine |
| Why do we need buffers for enzymes? | Because some enzymes only have optimal activity over a narrow pH range |
| What types of bonds between E and S would potentially be disrupted by a change in pH? | H bonds, electrostatic interactions |
| What happens when an H is removed, due to a change in pH? | No H-bond can be formed - disrupting interaction between E and S |
| What happens when an H is added, due to a change in pH? | An H-bond might form that is not usually formed - disrupting interaction between E and S |
| How does adding an H affect the electrostatic interaction? | Adding an H can turn COO- into COOH - therefore, no H-bond can be formed - losing potential electrostatic interaction |
| How does removing an H affect the electrostatic interaction | Remvoing an H can turn NH3 into NH2 - an H-bond might form - losing potential electro static interaction |
| What might happen to the overall structure of the enzyme with a change in pH? | Enzymes are proteins therefore can be denatured by a change in pH |
| How are enzymes denatured from a change in pH? What types of bonds within enzyme can be disrupted? | Tertiary and quaternary structure will be disrupted and may reform differently, changing the function |
| There is also a genetic component to enzyme expression so what can be said about the induction/repression of enzymes? | Induction or repression occurs at the level of the genes |
| What is an example of enzyme induction? | Lac Operon - DNA encodes for enzymes that digest lactose. In absence of another energy source, induction with lactose leads to transcription of DNA into mRNA encoding for enzymes that digest lactose |
| Explain how enzyme repression would work | When there is an excess of a certain product, it will inhibit transcription of genes that code for one or more enzymes in its production pathway - negative feedback control |
| What is covalent modification? | Involves altering the structure of an enzyme (or proenzyme) by making or breaking covalent bonds |
| What are the two types of covalent modification? | Reversible and irreversible |
| What is reversible covalent modification | Addition or removal of a group to enzyme that causes it to convert to its active or inactive form |
| What is a common group that can do reversible covalent modification? | Phosphate, methyl, acetyl group |
| Does addition of a group for reversible covalent modification activate or inactivate the enzyme? | It depends |
| What is irreversible covalent modification | Cleavage of peptide bonds in proenzymes or zymogens |
| What is the purpose of irreversible covalent modification? | Makes sure enzyme isn't used until it is at the correct location and when it's needed |
| Mobilization of glucose from glycogen involves phosphate groups- what type of covalent modification is this> | Reversible covalent modification |
| What type of covalent modification is the formation of active chymotrypsin? | Irreversible covalent modification |
| What is allosteric modification? | Uses binding to enzyme allosteric sites to change conformation and activity of the enzyme |
| What is allosteric modification typically used for? | To control regulatory enzymes |
| How many subunits do allosteric enzymes have? | More than one subunit - allosteric site is on one subunit, active site on another |
| What does it mean when an allosteric enzyme is an activator? | Increases binding of substrate to enzyme |
| What does it mean when an allosteric enzyme is an inhibitor? | Decreases binding of substrate to enzyme |
| What kind of shape is the substrate binding curve of an allosteric enzyme? | Sigmoidal |
| What is a homotropic allosteric effect? | Effector is the same as the substrate - positive or cooperative effect |
| What is a heterotropic allosteric effect? | Effector is different from the substrate - can be a positive of negative effect |
| What is an example of a homotropic allosteric effect? | Hemoglobin where oxygen is both |
| ATCase enzyme (aspartate transcarbamoylase) is allosterically regulated, what are the properties? | Catalyzes the committed step of CTP synthesis. Activated by ATP, inhibited by CTP |
| What does compartmentalization allow for? | Compartmentalization of enzymes via membrane-bound organelles allows for regulation by separation of enzymes from opposing pathways into different cellular compartments and selective transportation of substrates and creation of unique microenvironments |
| What is an example of compartmentalization for segregation of different pathways? | In the cell, the mitochondria deals with beta oxidation and the cytoplasm will do fatty acid synthesis |
| What is an example of compartmentalization for creating a microenvironment? | Lysosomes |
| Chymotrypsin is specific for cleaving aromatic AA's while elastase is specific for cleaving Gly/Ala AA's. While the active sites of both are similar, 2 Gly are found in the substrate binding region of chymotrypsin while it is Va/ Thr for elastase. Explain | Size plays a role. Small AA in binding region means bigger AA in pocket while big AA in binding region means smaller AA in pocket |
| What is the general rate equation for the simple reaction A→P? | Vo=--∆[A]/ ∆t = ∆[P]/∆t |
| What is Vo in the general rate equation for simple reactions? | Initial velocity - when [A] is so high that there is noreverse reaction of P → A |
| What is --∆[A]/ ∆t in the general rate equation for simple reactions? | Loss of reactant over time |
| What is ∆[P]/∆t in the general rate equation for simple reactions? | Appearance of product over time |
| What can --∆[A]/ ∆t = ∆[P]/∆t be converted to? | Vo=k[A]^x |
| Break down Vo=k[A]^x | Vo= rate; x= order of reaction; k=rate constant |
| What is a first order reaction? | Occurs when a single reactant is converted to a product - unimolecular reaction requiring no collisions |
| In a first order reaction, what happens when you double the [A}? | Doubles the rate |
| In a first order reaction, what happens when you half the [A}? | Halves the rate |
| What can you say about the relationship between the rate and concentration of the substrate at the beginning of the reaction in a first order reaction? | Rate and concentration is proportional |
| Why does the rate slow down at a higher [S] in a first order reaction? | Reaching saturation |
| What happens when reaction is zero order? | Fully saturated |
| What is a second order reaction? | Occurs when two molecules must collide for a reaction to occur |
| If you have two reactants whose orders is each 1, what is the overall order of the reaction? | 2 |
| With two reactants (A + B) with a first order reaction, what would happen if you doubled [A] but the [B] remained unchanged? | Double rate |
| With two reactants (A + B) with a first order reaction, what would happen if you doubled [B] but the [A] remained unchanged? | Double rate |
| With two reactants (A + B) with a first order reaction, what would happen if you doubled [A] and [B]? | Quadruple |
| What is the order of reaction in this equation 2A →P? | Rate: k[A]^2 therefore 2nd order reaction |
| In this reaction ( k[A]^2), what would happen to the rate if the concentration of the reactant was doubled? | Quadruple rate |
| In this reaction ( k[A]^2), what would happen to the rate if the concentration of the reactant was tripled? | 9x rate |
| What is a pseudo first order reaction? | When a reaction can actually be second order but experimentally looks like first order |
| How can a reaction be pseudo first order? | Double [A] doubles rate if [B] is unchanged but double [B] doesn't change rate |
| How is a pseudo first order reaction even possible? | There is excess of B so you always have enough to run reaction at full capacity but not necessarily enough of A to run at full capacity |
| What is a common reactant in a psuedo first order reaction? | Water |
| What is Michaelis-Menten kinetics used for? | Determine max rate of certain enzyme reactions and determine affinity between an enzyme and its substrate |
| In general, what type of reaction can Michaelis-Menten kinetics be used for? | First order at low substrate concentrations, zero order at high substrate concentrations |
| In the equation E + S → ES → E + P, what is k1, k2, k3, and k4? | k1= (E + S →ES); k2 =( E + S ← ES); k3 = (ES → E + P); k4 = (ES ← E + P) |
| Why can k4 be ignored during a reaction? | If the rates are measured when [P] is still very low |
| What can be inferred in the Michaelis-Menten kinetics about the rate? | Rate of ES formation = Rate of ES degradation |
| When would the rate of degradation = the rate of production formation? | If k2 is very small |
| If the original rate equation is ∆[P]/∆t, given the assumptions we have about the equation, what would be the new rate? | k3[ES] |
| Why is k3[ES] not the rate that is normally used to configure anything? | Not possible to easily measure [ES] so Michaelis and Menten derived a more useful expression |
| What is the Michaelis-Menten equation? | Vo=Vmax[S]/ ([S] + KM) |
| What is Km? | Michaelis constant - it is a measure of the affinity of an enzyme for a substrate |
| What is Km mathematically? | (k2+k3)/k1 → rate of ES degradation/rate of ES formation |
| If k1 is large, then Km is small - what is the implication of this? | Small Km indicates that enzyme has a high affinity for a substate |
| An AA substitution of Glu to Lys in enzymes increases Km of E for NAD+ from 300 micrometers to 7000 micrometers. What does this say about the binding of the NAD+ to the enzyme and why is there such a change? | The increase in Km indicates that there is a lower affinity and the small AA change is going from Glu (basic) to Lys (acidic) |
| Why is the Michaelis-Menten equation so useful? | Can determine affinity of an enzyme for a substrate by plotting rate against [S] |
| What happens to the substrate concentration when you half Vmax? | The substrate concentration is equal to Km - [S]=Km |
| What is the drawback to determining Km via this method? | Difficult to exactly experimentally assess Vmax |
| If it is difficult to experimentally assess Vmax, how can we determine Vmax and Km? | By taking reciprocal of the M&M equation. Vo=Vmax[S]/ ([S] + KM) → 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]) |
| In the equation, 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]), what is 1/Vo? | Y-axis |
| In the equation, 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]), what is 1/Vmax? | Y-intercept |
| In the equation, 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]), what is Km/Vmax | Slope |
| In the equation, In the equation, 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]), what is , what is 1/[S]? | X-axis |
| When plotting In the equation, 1/Vo= (1/Vmax) + (KM/Vmax)(1/[S]), what is the graph known as? | Lineweaver-Burk plot |
| What does the Lineweaver-Burk plot allow for? | Extrapolate values for both Vmax and Km |
| In a Lineweaver-Burk plot, how do you determine which has the best affinity between E and S? | You'd want the smallest Km so find the line which has the smallest X-intercept (-1/Km) so line furthest from y-axis |
| In a Lineweaver-Burk plot, how do you determine which has the highest Vmax? | Highest Vmax would use the y-intercept to determine (1/vmax) so line closest to x-axis |
| What can we use Km for? | Determine enzyme-substrate affinity, determine enzyme efficiency |
| What is the enzyme efficiency? | Ratio of two values: Kcat (measures speed of P formation once ES has been made) and Km |
| What is the turnover number? | Kcat - number of molecules of S converte to P over time with a fully saturated enzyme |
| What is the mathematical definition of Kcat? | Vmax/[Et] where Et is the total enzyme concentration |
| What gives a more efficient enzyme a large or small value for Kcat/Km? | Larger |
| T/F - Enzyme can work faster than the time it takes for substrate to diffuse into active site. | False - enzyme cannot work faster than time it takes for substrate to diffuse into active site |
| What is the diffuse control limit? | Minimum of 10^8-10^9M^-1s^-1 |
| What is catalytic perfection? | Enzymes that approach the diffusion control limit - every time a substrate reaches the active site, a product is formed |
| Michaelis-Menten kinetics can also lend itself to the study of enzyme inhibition. What are some applications of enzyme inhibition? | Physiological regulation of enzyme function and clinical therapies |
| What is an example of physiological regulation of enzyme function that Michaelis-Menten kinetics can be used for? | Classic example: P will inhibit production of further P |
| What is an example of clinical therapies of enzyme function that Michaelis-Menten kinetics can be used for? | HIV, AIDS treatment- drugs that target viral enzymes required to make new virus. Lexiva - protease inhibitor that preventsHIV1 protease from cutting long viral protein precursors into their useable form, preventing formation of new, active viral particles |
| What kind of enzyme inhibition follows Michaelis-Menten kinetics? | Reversible - competitive, uncompetitive, noncompetitive |
| What kind of enzyme inhibition does not follow Michaelis-Menten kinetics? | Irreversible and allosteric |
| What is competitive inhibition? | Reversible binding of the inhibitor to the active site of the enzyme |
| In competitive inhibition, does Vmax change? | No, it just takes more [S] to reach Vmax because there is an I present - S must "compete" with I. Structure and concentration of enzyme doesn't change |
| In competitive inhibition, what happens to Km? | There is an increase because there is a worse affinity for E and S due to the presence of I but the affinity for S isn't actually changing |
| Describe any changes to the slope, y and x intercept, and others in a competitive inhibition | Y-intercept stays the same but the slope and x-intercept changes |
| What is the implication of the Lineweaver-Burk plot in competitive inhibition? | Appears to fan out at y-intercept |
| What is Vmax? | Depends on structure of enzyme and [E]. Can allow to determine affinity of an enzyme for a substrate by plotting against [S] |
| What is uncompetitive inhibition? | Reversible binding of I to ES |
| In uncompetitive inhibition, does Vmax change? | Yes, it is lower because I interferes with S binding and decreases catalysis of ES in ES complex |
| In uncompetitive inhibition, what happens to Km? | It is lower because Vmax is lower - decrease in ES complex. |
| In uncompetitive inhibition, is the affinity for E for S actually changing? | According to Le Chatelier's principle, the decrease in ES complex will cause more free enzyme to convert to ES form to increase the amount of ES overall and this increase would infer that there is a a higher affinity (lower Km) |
| What is the shape of the Lineweaver-Burk plot in an uncompetitive inhibition? | Parallel to original - same slope, y-intercept changes |
| What is noncompetitive inhibition? | Reversible binding of I to E or ES |
| In noncompetitive inhibition, does Vmax change? | Yes, it is lower because I is still interfering with S binding and decrease in catalysis of ES in ES complex |
| In noncompetitive inhibition, what happens to Km? | It doesn't change. Cancels out because binding at both E and ES and increasing [S] won't overcome inhibitor because Vmax is lowered |
| What is the shape of the Lineweaver-Burk plot for noncompetitive inhibition? | Appears to fan out at x-intercept. Slope changes, y-intercept changes but not x-intercept |
| What is irreversible inhibition? | Occurs upon covalent binding of I to active site of E that's irreversible |
| What are some examples of irreversible inhibition? | Anemia seen in lead poisoning - Pb binds to sulfhydryl groups in enzymes involved in heme synthesis. Penicillin blocks enzymes required for synthesis of bacterial cell walls |
| How does penicillin block enzymes required for synthesis of bacterial cell walls? | Glycoprotein peptidase catalyzes bacterial cel wall synthesis but penicillin irreversibly inhibits this enzyme by forming a covalent bond with the Ser residue of the enzyme |
| In allosteric binding, what happens to the graph in the presence of an inhibitor molecule? | Graph will shift to the right because will need more substrate |
Created by:
nnguyen44
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