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Chapter Summary: pg. 66

Ch. 2 – Enzymes 2.1 Enzymes as Biological Catalysts 

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Catalysts: don’t impact the thermodynamics of a biological reaction o The DHrxn & eqbm position don’t change o Help the rxn proceed at a much faster rate Enzymes: biological catalysts o Don’t change during the course of the rxn o Increases the rxn rate of a process by a factor of 100, 1000, or even 1,000,000,000,000 (1012) times when compared to uncatalyzed reaction  W/o this increase, we wouldn’t be alive Key features of enzymes: 1. Lower the activation energy 2. Increase the rate of the reaction 3. Don’t alter the eqbm constant 4. Aren’t changed or consumed in the reaction (which means that they’ll appear in both the reactants & products) 5. Are pH- & temperature-sensitive, with optimal activity at specific pH ranges & temperatures 6. Don’t affect the overall DG of the reaction 7. Are specific for a particular reaction or class of reactions

Enzyme Classifications  Substrates: the molecules upon which an enzyme acts o Enzymes are picky  Enzyme specificity: a given enzyme will only catalyze a single reaction or class of reactions with these substrates  Urease only catalyzes the breakdown of urea  Chymotrypsin can cleave peptide bonds around the aa’s phenylalanine, tryptophan, and tyrosine in a variety of polypeptides o All these aa’s contain an aromatic ring, which makes chymotrypsin specific for a class of molecules  Most enzymes have descriptive names ending in suffix –ase o Lactase breaks down lactose  Can be classified into 6 categories, based on their function or mechanism: 1. Oxidoreductases: catalyze oxidation–reduction reactions (transfer of electrons b/w biological molecules)  Often have a cofactor that acts as an electron carrier (i.e., NAD+ or NADP+)  In rxns catalyzed by oxidoreductases:  Reductant: the electron donor  Oxidant: the electron acceptor  The convention for naming reductants & oxidants of oxidoreductases is the same as the convention for naming reducing agents & oxidizing agents in general & organic chem  Enzymes with dehydrogenase or reductase in their names are usually oxidoreductases  Enzymes in which oxygen is the final electron acceptor often include oxidase in their names 2. Transferases: catalyze the movement of a functional group from 1 molecule to another  Protein metabolism: an aminotransferase can convert aspartate & a -ketoglutarate (as a pair) to glutamate & oxaloacetate by moving the amino group from aspartate to a ketoglutarate  Most will be straightforwardly named  Kinases = a member of this class  Kinases: catalyze the transfer of a phosphate group, generally from ATP, to another molecule 3. Hydrolases: catalyze the breaking of a compound into 2 molecules using the addition of water  In common usage, many are named only for their substrate

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Common hydrolases:  Phosphatase: cleaves a phosphate group from another molecule o One of most common hydrolases encountered on MCAT  Peptidases: break down proteins  Nucleases: break down nucleic acids  Lipases: break down lipids 4. Lyases: catalyze the cleavage of a single molecule into 2 products  Don’t require water as a substrate  Don’t act as oxidoreductases  Bc most Es can also catalyze the reverse of their specific rxns, the synthesis of 2 molecules into a single molecule may also be catalyzed by a lyase  When fulfilling this function, it’s common to refer to them as synthases 5. Isomerases: catalyze the rearrangement of bonds within a molecule  Depending on the mechanism of the enzyme, some also classified as:  Oxidoreductases  Transferases  Lyases  Catalyze rxns b/w stereoisomers as well as constitutional isomers 6. Ligases: catalyze addition or synthesis reactions, generally b/w large similar molecules, and often require ATP  Synthesis rxns with smaller molecules are generally accomplished by lyases  Most likely to involve nucleic acid synthesis & repair on Test Day Impact on Activation Energy  Thermodynamics relates the relative energy states of a rxn in terms of its products & reactants o Endergonic reaction: requires energy input (DG > 0); takes in energy as it proceeds o Exergonic reaction: energy is given off (DG < 0); release energy as it proceeds

The activation energy req’d for a catalyzed rxn is lower than that of an uncatalyzed rxn while the D G (and D H) remain the same. This rxn is spontaneous; DG is negative.

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Enzymes DON’T: o Alter the overall free energy change for a rxn o Change the eqbm of a rxn Enzymes DO: o Affect the rate (kinetics) at which a rxn occurs Enzymes can affect how quickly a rxn gets to eqbm but not the actual eqbm itself Es ensure that many important rxns can occur in a reasonable amt of time in biological systems o *As catalysts, Es are unchanged by the rxn  Consequence: far fewer copies of the enzyme are req’d relative to the overall amt of substrate bc 1 enzyme can act on many, many molecules of substrate over time Catalysts exert their effect by lowering activation energy of a rxn o Make it easier for the substrate to reach the transition state Most rxns catalyzed by enzymes are technically reversible o Although that reversal may be extremely energetically unfavourable

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\ essentially nonexistent

2.2 Mechanisms of Enzyme Activity 

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Enzyme mechanisms vary depending on the rxn being catalyzed o But they do tend to share common features  Enzymes may act to: a) Provide a favourable microenv’t in terms of charge or pH b) Stabilize the transition state c) Bring reactive groups nearer to one another in the active site Key catalytic activity of the enzyme = the formation of the enzyme-substrate complex in the active site of an enzyme o Reduces the activation energy of the rxn Interaction b/w a substrate & active site of enzyme also accounts for the selectivity & some regulatory mechanisms of Es

Enzyme–Substrate Binding  Substrate: the molecule upon which an enzyme acts  Enzyme-substrate complex: the physical interaction b/w the substrate & the enzyme  Active site: the location within the enzyme where the substrate is held during the chemical reaction

Reaction Catalysis in the Active Site of an Enzyme This transferase has catalyzed the formation of a bond b/w 2 substrate molecules.

Assumes a defined spatial arrangement in the ES complex  Dictates the specificity of that enzyme for a molecule or group of molecules o The following interactions/bonds within the active site all stabilize this spatial arrangement & contribute to the efficiency of the enzyme: 1. Hydrogen bonding 2. Ionic interactions 3. Transient covalent bonds 2 competing theories explain how enzymes & substrates interact (1 of the 2 is better supported than the other) o

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1. Lock & Key Theory  Suggests that the enzyme’s active site (lock) is already in the appropriate conformation for the substrate (key) to bind  Substrate can then easily fit into the active site (like a key into a lock)  No alteration of the tertiary or quaternary structure is necessary upon binding of the substrate 2. Induced Fit Model (more scientifically accepted theory)  More likely to see this 1 referenced on MCAT  Starts w/ a substrate & an enzyme active site that don’t seem to fit together  Once the substrate is present & ready to interact with the active site, the molecules find that the induced form (transition state) is more comfortable for both of them  Shape of the active site becomes truly complementary only after the substrate begins binding to the enzyme o The substrate has induced a change in the shape of the enzyme  This interaction requires energy  this part of the rxn is endergonic  A substrate of the wrong type won’t cause the appropriate conformational shift in the enzyme  \ active site won’t be adequately exposed, the transition state isn’t preferred, and no rxn occurs Cofactors & Coenzymes  Cofactors or coenzymes: nonprotein molecules o Req’d by many enzymes to be effective o Tend to be small in size so they can bind to the active site of the enzyme & participate in the catalysis of the reaction  Usually by carrying charge through either: a) Ionization b) Protonation c) Deprotonation o Usually kept at low concentrations in cells – can be recruited only when needed  Apoenzymes: enzymes w/o their cofactors  Holoenzymes: enzymes containing their cofactors  Attached in variety of ways o Ranging from weak noncovalent interactions to strong covalent ones

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Prosthetic groups: tightly bound cofactors or coenzymes that are necessary for enzyme function Cofactors = generally inorganic molecules or metal ions; often ingested as dietary minerals Coenzymes = small organic groups, the vast majority of which are vitamins or derivatives of vitamins (i.e., NAD+, FAD, and coenzyme A) o 2 major classes of vitamins:  Water-soluble vitamins = B complex vitamins & ascorbic acid (vitamin C)  Important coenzymes that must be replenished regularly bc they’re easily excreted  B vitamins: o B1 – thiamine o B2 – riboflavin o B3 – niacin o B5 – pantothenic acid o B6 – pyridoxal phosphate o B7 – biotin o B9 – folic acid o B12 – cyanocobalamin o MCAT is unlikely to expect memorization of B vitamins; however, familiarity with their names may make biochem passages easier  Fat-soluble vitamins = A, D, E, and K  Better regulated by partition coefficients o Partition coefficients quantify the ability of a molecule to dissolve in a polar vs. nonpolar env’t Enzymatic rxns aren’t restricted to a single cofactor or coenzyme o Metabolic rxn often require magnesium, NAD+ (derived from vitamin B3), and biotin (vitamin B7) simultaneously Deficiencies in vitamin cofactors can result in devastating disease o Thiamine = essential cofactor for several Es involved in cellular metabolism & nerve conduction  Thiamine deficiency  Often a result of excess alcohol consumption & poor diet  Results in diseases including Wernicke-Korsakoff syndrome o Patients suffer from a variety of neurologic deficits, incl. delirium, balance problems, and (in severe cases) the inability to form new memories o

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2.3 Enzyme Kinetics 

V high yield

Kinetics of Monomeric Enzymes  Enzyme kinetics are dependent on factors like: 1. Env’tal conditions 2. Concentrations of substrate [S] & enzyme [E]  Greatly affect how quickly a rxn will occur  High [E] relative to [S] – many active sites available  Will quickly form products  Chemical sense, would reach eqbm quickly  As slowly add more substrate, the rate of the rxn will increase  Rxn rate can’t go any faster once it has reached saturation  Saturation: the point at which the enzyme is working at maximum velocity (vmax) o Only way to increase vmax is by increasing the [E]  In the cell, this can be accomplished by inducing the expression of the gene encoding the enzyme

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Michaelis–Menten Plot of Enzyme Kinetics As the amt of substrate increases, the enzyme is able to increase its rate of reaction until it reaches a maximum enzymatic reaction rate (vmax). Once vmax is reached, adding more substrate won’t increase the rate of reaction.

Michaelis–Menten Equation  For most Es, this eqn describes how the rate of the reaction ( v) depends on the concentration of both the enzyme [E] & the substrate [S], which forms product [P] o k1 = rate E-S complexes form o k-1 = rate E-S complexes dissociate o kcat = rate E-S complex turns into E + P (enzyme + product)

k1 E+S ⇌ ES k cat E+P → k −1 

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In either case, the enzyme is again available o On Test Day, [E] will be kept constant  Under these conditions, can relate the velocity of the enzyme to substrate concentration using the Michaelis-Menten eqn:

v max [ S] K m +[ S ] 

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When the rxn rate is equal to half of vmax, Km = [S]:

Km = Michaelis constant o The substrate concentration at which half of the enzyme’s active sites Can assess an E’s affinity for are full a S by noting the Km. o Often used to compare enzymes Low Km: reflects high affinity o Under certain conditions, Km = a measure of the affinity of the E for its for the S (low [S] req’d for S 50% E saturation)  When comparing 2 Es, the 1 with higher Km has the lower High Km: reflects low affinity affinity for its substrate of the E for the S  Bc it requires a higher [S] to be half-saturated o Km value = an intrinsic property of the E-S system  Can’t be altered by changing the concentration of substrate or enzyme  For a given [E], the Michaelis-Menten relationship generally graphs as a hyperbola (^) o When [S] < Km  changes in [S] will greatly affect the reaction rate o At high [S] exceeding Km  the reaction rate increases much more slowly as it approaches vmax, where it becomes independent of [S]  vmax = maximum enzyme velocity; measured in moles of enzyme per second o Can be mathematically related to kcat (units of s–1)

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v max =[ E ] k cat 

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v= 

v=

Qualitatively, kcat measures the # of substrate molecules “turned over” (converted to product) per enzyme molecules per second  Most Es have kcat values b/w 101 & 103 Michaelis-Menten eqn can be restated using kcat:

k cat [ E ] [ S ] K m +[ S ] At v low [S], where Km >> [S]:

k cat [E ] [ S] Km o

Ratio kcat/Km = catalytic efficiency of the enzyme  A large kcat (high turnover) or a small Km (high substrate affinity) higher catalytic efficiency (indicates a more efficient enzyme)

k1

Rate E-S complexes form

k-1

Rate E-S complexes dissociate

Km

Michaelis constant The [S] at which ½ E’s active sites are full A measure of affinity of E for its S (under certain conditions)

kcat (s-1)

vmax

Rate E-S complex turns into E + P Measures the # of S molecules “turned over” (converted to product) per E molecules per second Maximum E velocity; moles of E/s

Lineweaver-Burk Plots  A double reciprocal graph of the Michaelis–Menten equation o The same data graphed in this way yield a straight line  The actual data are represented by portion of the graph to the right of the y-axis o But the line is extrapolated into the upper left quadrant to determine its intercept with the x-axis

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x-intercept  value of

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y-intercept  value of

−1 Km 1 v max

Especially useful when determining the type of inhibition that an E is experiencing bc vmax & Km can be compared w/o estimation

Cooperativity  Certain Es don’t show normal hyperbola when graphed on a Michaelis-Menten plot ( v vs. [S]), but rather show sigmoidal (S-shaped) kinetics o Owing to cooperativity among substrate binding sites  Cooperative enzymes have multiple subunits & multiple active sites o Subunits & enzymes may exist in 1 of 2 states: 1. Low-affinity tense state (T) 2. High-affinity relaxed state (R) o Binding of the S encourages the transition of other subunits from the T state to the R state

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 Increases the likelihood of S binding by these other subunits Loss of S can encourage transition from R state to T state  Promotes dissociation of substrate from the remaining subunits Enzymes showing cooperative kinetics are often regulatory enzymes in pathways o Phosphofructokinase-1 in glycolysis Cooperative Es = subject to activation & inhibition, both competitively & through allosteric sites Cooperativity can be quantified using Hill’s coefficient (numerical value) o Value of Hill’s coefficient indicates the nature of binding by the molecule:  Hill’s coefficient > 1: positively cooperative binding is occurring  After 1 ligand is bound the affinity of the enzyme for further ligand(s) increases  Hill’s coefficient < 1: negatively cooperative binding is occurring  After 1 ligand is bound the affinity of the enzyme for further ligand(s) decreases  Hill’s coefficient = 1: the enzyme doesn’t exhibit cooperative binding o

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2.4 Effects of Local Conditions on Enzyme Activity 

Activity of an enzyme is heavily influenced by its env’t o In particular, the following have significant effects on the ability of an enzyme to carry out its function: 1. Temperature 2. Acidity or alkalinity (pH) 3. High salinity o On the MCAT, the terms enzyme activity, enzyme velocity, and enzyme rate are all used synonymously

1. Temperature o Enzyme-catalyzed reactions tend to double in velocity for every 10°C increase in temperature until the optimum temperature is reached  For the human body, this is 37°C (98.6°F or 310 K)  After this, activity falls off sharply – the E will denature at higher temperatures  Some Es that are overheated may regain their function if cooled o Real-life example of temperature dependence  Siamese cats  Siamese cats are dark on their faces, ears, tails, and feet but white elsewhere  Enzyme responsible for pigmentation (tyrosinase) is mutated in Siamese cats o Ineffective at body temp but at cooler temps becomes active  \ only the tail, feet, ears, and face (cooled by air passing through the nose & mouth) have an active form of the enzyme & are dark 2. pH o

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Most enzymes also depend on pH to function properly  pH affects the ionization of the active site  Changes in pH can lead to denaturation of the enzyme For Es that circulate & function in human blood, optimal pH is 7.4

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pH < 7.35 in human blood = acidemia

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Even though it’s more basic than chemically neutral 7.4, it’s more acidic than physiologically neutral 7.4 Exceptions to pH 7.4:  Both in digestive tract o Pepsin: works in the stomach; has a maximal activity around pH 2 o Pancreatic enzymes: work in the small intestine; work best around pH 8.5

3. Salinity o Effect of salinity or osmolarity isn’t generally of physiologic significance, BUT altering the concentration of salt can change enzyme activity in vitro o Increasing levels of salt can disrupt hydrogen & ionic bonds, causing a partial change in the conformation of the enzymes  In some cases causing denaturation

2.5 Regulation of Enzyme Activity 

Body must be able to ctrl when Es work o Enzymes involved in mitosis should be shut off when cells are no longer dividing (in the G0 phase)

1. Feedback Regulation o Feedback regulation: process in which enzymes are subject to regulation by products further down a given metabolic pathway; more often o Feedforward regulation: process in which enzymes are regulated by intermediates that precede the enzyme in the pathway; less often o Some examples of feedback activation, but feedback inhibition is far more common  Negative feedback (feedback inhibition)  Helps maintain homeostasis o Once we have enough of a given product, we want to turn off the pathway that creates that product, rather than creating more o Product may bind to the active site of an enzyme or multiple enzymes that acted earlier in its biosynthetic pathway  Competitively inhibiting these Es & making them unavailable for use

2. Reversible Inhibition o 4 types: a) Competitive Inhibition  Occupancy of the active site o Substrates can’t access enzymatic binding sites it there’s an inhibitor in the way  Can be overcome by adding more substrate so that the substrate-to-inhibitor ratio is higher  If more molecules of substrate are available than molecules of inhibitor, then the E will be more likely to bind substrate than inhibitor Lineweaver-Burk Plot o Assuming the enzyme has equal affinity for both molecules  Adding a competitive inhibitor has the follow effects on various measures: i. vmax – no effect  Bc if enough substrate is added, it will outcompete the inhibitor & be able ...