Biochemistry Lecture Four

Front 1

Enzyme

Back 1

- enzymes are proteins or nucleic acid catalysts which speed the rate of biochemical reactions
- highly specific
- high sensitivity to change in temp, pH, substrate concentration, cofactor availability and ionic environment

Front 2

Enzyme catalysis

Back 2

- occurs after the substrate (S) binds to the active side of the enzyme (E) to form an enzyme-substrate complex "ES". The Enzyme-substrate complex undergoes a reaction or catalytic alteration to form the product (P) and reform the enzyme
- the enzyme is unchanged in the process and free to participate in another round of catalysis
- key to enzyme catalysis is that rxn pathway and energy level of transition state are altered by interactions between the substrate and the active site AA side chains.

Front 3

Active site of enzyme

Back 3

- pocket, crevice or niche on the surface of a protein, which is comprised of AA side chains from various parts of the primary sequence, it is where the substrate binds. In other words, critical groups facilitate catalysis
- specificity depends on the geometric and chemical complementarity of the substrate surface and the surface of the active site of the enzyme (AA residues)
- two features of enzyme activity are binding and catalysis
- substrate is bound to the active site by weak interactions with the side chains of the enzyme
- interactions between enzyme and substrate include ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces

Front 4

What are the types of possible reactions between the substrate and the active site AA side chains

Back 4

- acid-base catalysis
- nucleophilic and electrophylic interactions
- hydrophobic effects
- ionic stabilization of charged intermediates
- formation of covalent bond between the substrate and enzyme

Front 5

What are the two hypothesis used to explain substrate binding

Back 5

-lock and key
-induced fit
- both hypothesis have some validity. All enzymes undergo some changes in active site topography when the substrate is bound, but the enzymes vary considerably in the degree of change that occurs upon substrate binding.

Front 6

What is the lock and key hypothesis

Back 6

- substrate fits immediately into the active site like a key in a lock
- suggests that the shape of active site is the same before and after the substrate is bound

Front 7

Induced fit hypothesis

Back 7

- shape of active site changes in the presence of the substrate to yield a precise fit
- interactions between substrate and active site AA induce conformational changes in the shape of the active site.

Front 8

Hexokinase

Back 8

- example of induced fit
- active site of hexokinase undergoes a significant alteration (cleft closes around the substrate) in its conformation when its substrate, glucose is bound.

Front 9

effect of temp on enzyme activity

Back 9

- enzyme reactions are generally more sensitive to changes in temp than chemical rxns are
- rate of rxn increases as temp is increased
-sensitivity to temp measured in terms of Q10
- enzymes subject to inhibition at higher temps...enzyme activity increases with temp up to some optimal temp range, but then decreases as the temp is raised even higher. Above a certain temp, enzymes lose activity because they are irreversibly denatured

Front 10

Q10

Back 10

- the increase in enzyme activity for a 10 degree rise in temp
- Q10 is generally higher for enzyme reactions than it is for chemical reactions

Front 11

pH affect on enzymes

Back 11

- most enzymes sensitive to pH
- pH range in which optimal activity is observed, at pH values below or above this value, enzyme activity decreases, and at pH extremes, most enzymes are denatured.

Front 12

Why is the effect on pH more pronounced than it is in chemical reactions?

Back 12

- In addition to its effects on substrate, the pH influences the shape and charge of the active site, which in turn changes the functionality of the enzyme in catalysis.

Front 13

Describe the catalytic activity of an enzyme

Back 13

- ability to convert substrate (S) to product (P)
- measured as micro-moles of product/minute (enzyme units)
- under optimal conditions 1U= 1micro-mole product/min

Front 14

Specific activity

Back 14

- ratio of enzyme activity to the amount of protein present
- measured as Enzyme Units/mg of protein
- specific activity is generally low in crude biological extracts and increases as the enzyme is purified

Front 15

What is an indication that an enzyme preparation has reached homogenity?

Back 15

- specific activity does not increase with further purification procedures are attempted

Front 16

Cofactor

Back 16

-often required for enzyme reactions (so catalysis would require the enzyme, substrate and that cofactor)
- can be organic molecules or inorganic ions

Front 17

apoenzyme

Back 17

- enzyme that lacks an essential cofactor

Front 18

holoenzyme

Back 18

- enzyme with a cofactor bound

Front 19

organic cofactors

Back 19

- commonly participate in oxidation-reduction reactions and in the transfer of various organic functional groups
-often vitamins or contain vitamin components

Front 20

inorganic cofactors

Back 20

participate in oxidation-reduction reactions, substrate binding and may also facilitate the correct active conformation of a protein
-

Front 21

Describe the rate of enzyme reactions and uncatalyzed reactions

Back 21

-catalyzed reactions rapid at first (speed of product formation), but drop quickly as substrate levels decrease and the reaction approaches equilibrium
- uncatalyzed reactions are very slow
- both catalyzed and uncatalyzed reactions approach the same equilibrium value, just at different speeds.
**enzymes can't change the equiliburim of the reaction, they only influence the rate of the chemical reaction. The rate of the forward and reverse reactions are enhanced to the same degree. Enzymes accelerate the attainment of equibira but do not shift their position

Front 22

Turnover number of rxn

Back 22

Kcat = Vmax/Et
- number of molecules of substrate that can be converted per second per molecule of enzyme (or per enzyme active site for multi-subunit enzyme)
- relate the the molecules of a specific enzyme not the total protein in a preparation.
**unlike specific activity, which increases as an enzyme is purified, the turnover number of an enzyme is an intrinsic property which does not change with purficiation

Front 23

free energy of a reaction

Back 23

- amount of energy released or consumed by a reaction (delta G)
- difference in energy levels between the substrate and product (G final state minus G initial)
- thermodynamic factor dependent on levels of the reaction substrate and product
- an enzyme doesn't change these levels and thus doesn't change the overall free energy of a reaction

Front 24

standard free energy

Back 24

-delta G ^0
- measures the energy released of consumed by a reaction when all substrates are present at 1M concentrations under standard conditions or temperature and pressure

Front 25

describe a thermodynamically favorable reaction

Back 25

- delta G is negative
- energy releasing rxn
- at equilibrium there will be more product than substrate

Front 26

free energy of activation

Back 26

- difference between the energy level of a substrate and the activated intermediate or "transition state" of the reaction.
- an enzyme can provide an alternate transition state for a reaction and thus can change the free energy of activation of a reaction

Front 27

Describe a reaction when the product and the substrate are equal

Back 27

- delta G is zero
- at equilibrium there is no difference in relative concentrations of substrate and product

Front 28

describe a thermodynamically unfavorable reaction

Back 28

- product is at a higher energy level than the substrate
-transformation from substrate to product requires energy
- at equilibirum there will be more substrate than product

Front 29

What does an enzyme change in a rxn

Back 29

- changes the energy of activation but does not change the free energy of the rxn and does not change the eqilibrium.
- Enzymes do NOT change the thermodynamics of a rxn, meaning they do nto change the delta G, the equilibirum constant (Keq), the amount of free energy released or required or the net balance of substrate and product at equilibrium

Front 30

Explain why delta G^0 is a problem for biochemists

Back 30

- a hydrogen ion level of 1M is equivalent to pH0 and biochemical reactions generally take place at or around pH7
- so a modification of standard free energy is used, the prime ' denotes that the H+ concentration is set at pH 7 (10^-7) instead of at 1M

Front 31

How is the equlibrium constant related to the standard free energy

Back 31

- at equilibrium, delta G = 0 and delta G prime = -RTlnK'eq where K'eq is the equilibrium constant for the rxn.
**equilibrium constant of rxn is related to the standard free energy, and neither can be changed by the presence of an enzyme, both are determined by the energy levels of the substrate and product
- if delta G^0' is negative K'eq will be a positive number greater than one
- if delta G^0' is zero K'eq will be one
- if delta G^0' is positive K'eq will be a positive number less than one

Front 32

What is Vo

Back 32

- initial reaction rate of an enzyme reaction, taken by measuring the enzyme rates as close to time zero as possible
- increases with increasing substrate levels
- when the initial rates of the enzyme reaction (Vo) are plotted against the substrate concentration, a hyperbolic plot is obtained

Front 33

Michaelis-Menton equation

Back 33

V=Vmax*(S)/(S)+Km
- derived for an enzyme reaction under steady state conditions (rate of reaction is constant
- assumed that the concentration of ES is constant during the rxn
- also assumed that there is essentially no product around, a condition that allows one to ignore the reverse reactionin whtih P+E revert to ES
-

Front 34

Km

Back 34

Km= michaelis constant
Km=K2+K-1/K1
- Km gives an indication of how efficient an enzyme is at low (S)
- low Km tells us that an enzyme is efficient at low (S)
- high Km tells is that an enzyme is not efficient at low (S)
- When (S)=Km, then V=1/2*Vmax

Front 35

K1

Back 35

rate constant for the reversion of ES to E+S

Front 36

K2

Back 36

rate constant for the conversion of ES to E+P
- when K2 is rate limiting, the Km is an approximate measure of the affinity of E for S and Km is equal to the dissociation constant for the ES complex into E+S

Front 37

Dissociation Constant of ES Complex

Back 37

(S) at which v=Vmax/2

Front 38

Describe the effect of the substrate concentration on the reaction rate

Back 38

- at low substrate(S<Km), the M-M equation reduces to a straight line equation V=K(S)
- at a slightly higher substrate level, the reaction rate curves off and the response to substrate levels can't be approximated by a linear equation and the whole M-M eq must be used
- at very high substrate levels (S>Km), the reaction rate approaches Vmax as a limit. At that point, the rate is not significantly increased by raising the (S) to even higher levels

Front 39

Enzymes and kinetic efficiency

Back 39

- very diverse
- turnover numbers range from 40,000,000 per second to .5 per second
-Km(michaelis constants) also vary; most enzymes have Km values between 1mM and 1microM, but some Kms are as high as 1M (e.g. catalase)
- generally a high Km would indicate an inefficient enzyme, but catalase has turnover number of 40,000,000 so it can still function efficiently with a poor Km

Front 40

Lineweaver-Burk plot

Back 40

- an alternative method of plotting kinetic data
- derived by inverting the M-M equation
= 1/V=1/Vmax +Km/Vmax(S)
- plot of 1/V vs 1/S rather than V versus S
- advantage is that it gives a straight line, which aids in extrapolation from experimental data and allows an accurate determination of Vmax and Km
-intercept on plot on ordinate = 1/Vmax and intercept on abscissa = 1/Km.
- slope of line = Km/Vmax

Front 41

What is a chemical inhibitor

Back 41

- chemical inhibitors slow or prevent enzyme activity
- biochemical reagents, toxins, venoms, antibiotics or drugs
- the type of interference can provide information about the mode of enzyme action
- irreversible, reversible and competitive and non-competitive

Front 42

competitive inhibitor

Back 42

- often look like an enzyme's substrate (or one of its transition state intermediates) and inhibit the enzyme by competing for occupancy of the active site
- methotrexate is one example and is used as a chemotherapy treatment.. It resembles dihydrofolate and inhibits enzymes that use dihydrofolate as a cofactor.
- bind reversibly to the active site of an enzyme; can be displaced by the substrate.
- competitive inhibition can be completely overcome by increasing the substrate concentration to high levels
- raise the Km but do not affect Vmax

Front 43

Describe the lineweaver-burk plot in the absence and presence of a competitive inhibitor

Back 43

- 1/V intercept remains the same in the absence of presence of the inhibitor, indicating that the Vmax does not change
- the 1/(S) intercept, which gives -1/Km becomes less negative, indicating that the apparent Km increases in the presence of competitive inhibitor
*** this is a double reciprocal plot, so the inhibited reaction plot is higher than the uninhibited reaction plot

Front 44

Uncompetitive inhibitor

Back 44

- bind reversibly only to the ES complex....the prior binding of the substrate to the enzyme is a prerequisite for the binding of the I to the ES
- a portion of the ES-complex is diverted to form a non-productive ESI complex, which is unreactive in the formation of product
- Vmax can't be achieved even at high substrate concentrations
- Km value is lowered and becomes smaller as more inhibitor is dded

Front 45

Lineweaver-Burk plot of uncompetitive inhibition

Back 45

- slope of double-reciprocal plot does not change in the presence of inhibitor
- Both Vmax and Km are reduced by equivalent amounts
- maximal rate is reduced but it is approached at a lower substrate concentration
- because this is a double reciprocal plot, the inhibited reaction line is higher than the uninhibited line

Front 46

Noncompetitive inhibitors

Back 46

- bind reversibly away from the active site to reduce or prevent catalytic activity
- substrate can still bind, but the ESI complex does not proceed to form product
- can't be overcome by raising the substrate level because the substrate does not displace the inhibitor
- lowers Vmax but does not affect the Km

Front 47

Describe the Lineweaver-burk plot of noncompetitive inhibition

Back 47

- shows that the I/(S) intercept is not changed, thus the Km value is not altered
- 1/V intercept is raised showing that the Vmax decreased
- inhibited line is higher than uninhibited line

Front 48

Irreversible inhibitors

Back 48

- stick to an enzyme covalently and can't be easily removed from the enzyme by mild techniques such as dialysis
- e.g. TPCK

Front 49

TPCK

Back 49

- irreversible inhibitor
- analog of natural substrates for the enzyme chymotrypsin
- also an affinity label (reactive substrate analog)
- binds at active site of chymotrypsin and reacts irreversibly with a histidine residue in the active site of the enzyme

Front 50

DIFP

Back 50

- group specific irreversible inhibitor
- specifically modifies unusually active serine residues in the active site of serine proteases such as chymotrypsin
- also modifies the active site serine residue of acetylcholinesterase

Front 51

iodoacetamide

Back 51

- also an irreversible inhibitor
- reacts with activated cysteine residues in active sites of various enzymes
- acetamide portion of the inhibitor molecule forms a covalent bound with the sulfur atom in the active site of these enzymes

Front 52

six basic types of enzyme reactions

Back 52

- oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases
- all biochemical reactions fall into one of these six categories.