M2C: Enzyme Kinetics

Front 1

Ligand

Back 1

Any molecule bound to a protein

Front 2

Substrate

Back 2

Specific ligand(s) bound to an enzyme that the enzyme is supposed to catalytically convert to a product.

Front 3

Carriers

Back 3

Hemoglobin (O2), transferrin (Fe++), albumin (fatty acids)

Front 4

Receptors

Back 4

Membrane bound (for hormones and other signals), immunoglobulins (for antigens), enzyme regulatory subunits (for regulatory metabolites)

Front 5

Enzymes

Back 5

Resemble receptors and carriers but chemically modify ligand (catalysis). Most common type of protein (70% of known proteins)

Front 6

Units of dissociation constants

Back 6

always in molalrity (micromolar or minimolar)

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Kd

Back 7

Dissociation constant. The affinity an enzyme has for the ligand.
Kd = [L][E]/[LE]

Front 8

Ks

Back 8

Affinity for the substrate. A dissociation constant.
Ks = [S][E]/[ES]

Front 9

Ki

Back 9

Affinity for an inhibitor. A dissociation constant.
Ki = [i][E]/[iE]

Front 10

Km

Back 10

A semi-dissociation constant. Affinity for the substrate. Defines the substrate concentration for 50% binding of the substrate or 50% action of the enzyme. Defined by the M-M equation.
Km = [S]({Kcat[E]/Kobs}-1)
Km = [S]({Vmax/v}-1)

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Why these affinity constants are important

Back 11

For a drug to be effective, it must compete with the regular substrate. Therefore its affinity constant (Ki) must complete with the enzyme's affinity for the regular substrate (Km).

Front 12

Dissociation constants and the 2-log rule

Back 12

Km and Ki signify the mid-point of the binding curve. The 2-log rule applies in this case as the affinities for substrate of enzymes are rarely variable. At a one log (or ten-fold) increase in concentration of the substrate, almost all of the enzyme will be loaded and active. At a ten-fold (or one log) decrease in the concentration of substrate, most enzymes will be empty. This is a hundred-fold total difference.

Front 13

Enzymes' affinity for their substrate

Back 13

The current "normal" concentrations of metabolites is controlled to be appropriate for the enzymes that use them. Also the enzymes have evolved to bind substrates that work well for their function and for the available concentration of the substrate.

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Proper dosage of protein drugs

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Drugs must be within the correct range to appropriately bind whatever enzyme is the target (Ki). Too low a concentration will ineffectively compete with the substrate (Ks). Too high a concentration (>10xKi), however, will saturate the system and cause inhibition or other effect on other enzymes or receptors. This is called a "side effect". Most harmful consequences of clinical drugs are caused by these unintended side effects.

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Metabolism

Back 15

Overall set of chemical reactions within a cell or organism.

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Allosteric

Back 16

"Other form". Means an enzyme that is regulated by conformational change between active and inactive forms.

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Zygomen

Back 17

Precursor form of an enzyme that is only activated by proteolysis.

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Enzyme binding and naming

Back 18

Most enzymes bind two substrates. A donor substrate is often a general molecule that is widely used in biosynthesis. The specific acceptor substrate is the molecule to which the enzyme will add moiety. Enzymes are often named for this acceptor substrate and the type of transfer.

Front 19

Enzyme naming

Back 19

Enzymes named for:
1. substrate + catalytic action + ase
ex. gluco + kin + ase
ribonucleotide reduct ase
2. Genes
ex. src, onc, ras
3. Size of protien
ex. p53, gp42, pp60
p = protein
gp = glycoprotein
pp = phosphoprotein

There are a few exception: pepsin, pepain, trypsin
named before creation of common naming trends.

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Metal cofactors

Back 20

More than 50% of enzymes use metal (ions) cofactors. Zn++, Fe++, Fe+++, Mg++, Cu++, K+, Mn++

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Coenzyme

Back 21

Complex organic cofactor

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Enzyme Control

Back 22

1. Enzymes react with only very specific substrates to create specific products.
2. Enzymes do not change the Keq for a reaction.
3. Enzymes increase the rate of the reaction by up to 10^17 (usually 10^5-10^9).

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Ubiquity of enzyme activity

Back 23

Every chemical reaction in the human body uses at least one enzyme.

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Active site/catalytic site

Back 24

Site on enzyme where substrate binds.

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Enzyme action

Back 25

Usually use induced fit to physically put substrate into position for faster rxn.

Front 26

Enzymes and free energy of activation

Back 26

Enzymes lower the free energy of reaction activation. This is the key to enhancing catalytic rates.

Front 27

Enzymes and pH

Back 27

Most humanenzymes function best at around 7 (pH of cells). Some have specific pHs though at which their activity is optimal. Usually has to do with environment. For example, pepsin in the stomach works best in an acidic environment. A dramatic change away from the optimal pH of an enzyme can reduce activity or render enzyme inactive.

Front 28

Enzymes and temperature

Back 28

Enzymes are effected by temp, but only at lower temps than body temp (37C). Higher temps increase non-catylized rxns, but at high temps, enzymes can be denatured. The lowest temp that works for enzymes in the body is the lowest temps the body can tolerate.

Front 29

Evaluation of Enzyme activity

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We are interested in the affinity of enzyme to their substrate and the velocity of action (v). The affinity is quantified by Km, the Michaelis-Menten constant.

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Ki

Back 30

If Ki for some inhibitor is low it has high affinity, if Ki is high, low affinity.

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Amount of product formed depends on...

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1. Time. More time = more product as long as enzyme and substrate concentrations are available.
2. Enzyme conc. More enzyme = more product.
3. Substrate conc. If substrate conc is high, will cause product until amount of product is sufficient and rxn stops.

Front 32

Binding constants

Back 32

Association constant: Ka, Dissociation constant: Kd
Substrate: Ks
Inhibitor: Ki
Unspecified ligand: KL

Always have units in molarity (micro or minimolar)

Front 33

Kd

Back 33

Affinity for any ligand.
Kd = [L][E]/[LE]

Front 34

Ks

Back 34

Affinity for any substrate.
Ks = [S][E]/[SE]

Front 35

Ki

Back 35

Affinity for any inhibitor.
Ki = [i][E]/[ES]

Front 36

Km

Back 36

Affinity for the substrate. Concentration of substrate when enzyme activity and/or binding is 50%.
Km = [S]({Kcat[E]/Kobs}-1)
Km = [S]({Vmax/v}-1)

Front 37

Drug affinity for enzyme

Back 37

In order for a drug (Ki) to be effective, must compete with substrate (Km) for binding with the target enzyme.

Front 38

Affinity constants and the 2-log rule

Back 38

Km and Ki represent the midpoint of a binding curve. In general, when the affinity is constant (as it is for most human enzymes), the range of enzyme activity ranges of 2-logs or 100-fold. From the mid-point, the addition of one log or 10 fold concentration of the substrate would cause almost all enzyme to be full and active. The ten fold or one log decrease in substrate concentration would cause most enzyme to be empty and inactive.

Front 39

Substrate and Enzyme Concentrations

Back 39

Most current metabolites are normally found in levels appropriate to the enzyme that processes them. Enzymes have evolved to interact with substrates that best fit their function and have adjusted to be at the correct concentrations for this substrate.

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Side Effects and Drug concentrations

Back 40

Drugs must be dosed specifically to target the enzyme they wish to inhibit. Too little will be insufficient to compete with substrate. Too much will cause likelihood of effecting/inhibiting other enzymes. This can cause "side effects". Most harmful consequences of clinical drugs are caused by these unintentional effects.

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Velocity

Back 41

v. The velocity or rate of a reaction. Units are usually nmol/min.

Vo =initial velocity

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k and kx

Back 42

rate of a single step in an overall reaction. Normal units are s^-1 (reciprocal seconds).

kx is the rate of a single step in a reaction sequence. Ex. k1, k2.

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kcat

Back 43

"Turnover number". Kcat = Vmax. Rate at which catalytic step of reaction takes place. Where actual chemistry occurs. Usually the slowest and therefore the absolute rate determining step.

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Lineweaver-Burk Plots

Back 44

Mathematical conversion of M-M plot (hyperbolic) gives a linear plot. Intercept on the y-axis to determine Vmax and intercept on x-axis to determine Km

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Efficiency

Back 45

Kcat/Km. Substrate has to find balance between fast and specific. If too specific (Km is low), the affinity for substrate is high and therefore the affinity for product is high and release is slower. If speed is too high (low Kcat), specificity is low.

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Normal Values for Kcat/Km

Back 46

Kcat is usually 10-10^3sec-1. If reaction must be very exact, Kcat will be slower. Values of 5x10^8-9M-1s-1 is near the upper limit of efficiency. For most enzymes, efficiency is around 10^-5 to 10^-7M-1s-1.

Front 47

Normal cellular concentrations of enzymes and metabolites

Back 47

Km is normally > or equal to [S]. Normal important substrates have conc. of 0.1-5mM (aa, carbs, nucleotides). Others are less abundant. Average conc. of all protein is ~1micromolar. [E] varies greatly, but in general:
low flux step: [E] = ~10-100nM
high flux step: [E] = 1-10micromolar

Front 48

Enzyme inhibition

Back 48

Inhibitors are usually analogs to the substrate and bind in the same place. Can be competitive or non-competitive (uncompetitive also exists). Ki only refers to reversible rxns. Covalent inhibition is irreversible.

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Competitive inhibitors

Back 49

Bind to same place as substrate and stop regular rxn from occurring. They can also add to another binding site, conformationally changing and de-activating enzyme. Competitive inhibitors do not actually change the affinity of enzyme for substrate, but do make Km ([S] for 50% bound) higher.

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Non-competitive inhibitors

Back 50

Inhibitor binds to enzyme so that substrate can still bind, but not well or enzyme is not as functional.

Front 51

Ki and competition

Back 51

Competition depends on affinities and concentrations of I and S. If Ki is less than Km, significant competition will occur. If [I] is close to Ki, significant inhibition will occur. Conc. of I must be half of conc. of E in order for Ki to occur.

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Tight-binding inhibitors

Back 52

If inhibitor is analog enough to substrate to stabilize the transition state of the enzyme, release will be slow and inhibition will be good.

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Non-competitive inhibitors are better

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In competitive inhibitors, substrate will build up due to non-processing, and will then out-compete inhibitor. Non-competitive is better.

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Suicide Inhibitors

Back 54

Covalently bond to enzyme making it inactive and unusable forever. This is good for drugs because by reducing the number of usable enzymes, drug dose can be reduced.