Ch 19

Ch 19. Enzymes and Vitamins

I. Enzymes

A. Catalysts in Metabolic Reactions

Ex. Oxidation of Glucose

C₆H₁₂O₆ + 6 O₂ --> 6 CO₂ + 6 H₂O + Energy DG = -2870 kJ/mol

Glucose- Thermodynamic Potentiality- has the ability to release stored chemical energy.

** Enzymes exert kinetic control over Thermodynamic potentiality **

- This means that enzymes control the rates at which certain reactions release their energy.

B. Features of Enzymes

1. Catalytic Power- acceleration of reaction rates (as much as 10¹⁶ times)

catalytic power = catalyzed rate

uncatalyzed rate

ex. Urease: hydrolysis of urea

catalyzed rate = 3 x 10⁴ rxns/sec uncatalyzed rate = 3 x 10^-10 rxns/sec

catalytic power is 1 x 10¹⁴. This means the enzyme is capable of increasing the reaction by 10¹⁴ times

a. turnover rate- the number of enzymes reactions per time

Resource: Catalytic power table

2. Specificity- Enzymes are highly selective for the substance they react on and the reaction they catalyze

a. substrate- the substance that an enzyme reacts on . These interact through intermolecular attractions (IMAs)

b. active site- the specific location on the enzyme where a substrate attaches.

c. "lock & key" model- the active site is selective for only one substrate with the specific geometric and

stereospecific structures

d. induced fit model- substrates are able to induce the active site conformation based on IMAs. -image

Resource: How does an enzyme work video

3. Regulation- an enzymes activity is regulated by

a. enzyme expression by DNA- (ex. lac repressors)

b. inhibitor and activator molecules

4. Reactions occur under milder conditions. Many enzymatic reactions occur under conditions that support

life (<100 ^oC and 1 atm). Reactions that are not catalyzed with enzymes require conditions not suitable for life.

C. Nomenclature

-most enzymes are commonly named for the substrate and name ending changed to -ase

Enzyme Classification- (NC-IUBMB) -- Enzyme Classification 2

1. International Commission on Enzymes - 1956

-set up a numbering system based on 6 classes of enzymes (now 7 classes-translocases). These are the Enzyme Commission Numbers

- all enzymes will be assigned a name beginning with E.C. and followed by 4 numbers

- the number are arranged in order of specificity. The class number arranges the enzyme by general function.

the second number usually allocates to what substrate, third confers either coenzyme or acceptor molecule and

the last identifies the specific enzyme by name.

ex. The 1^st reaction in Glycolysis is catalyzed by an enzyme that is specific to a 6-carbon sugar (e.g. glucose)

ATP + D-glucose --> ADP + D-glucose-6-phosphate

The classification of this enzyme is:

E.C. 2. transferase (transfering an atom/group of atoms to glucose)

E.C.2.7. transferase involving P-containing groups (the group contains phosphorus)

E.C.2.7.1. alcohol is a receptor (the receiving site on glucose is a hydroxyl)

E.C.2.7.1.1 6-carbon sugar (glucose is a 6-carbon sugar)

E.C.2.7.1.2 6-carbon sugar is glucose (specific to glucose sugar)

E.C.2.7.1.1 is called Hexokinase E.C.2.7.1.2- Glucokinase

Kinase is used for any ATP-dependent phosphotransferase

D. Coenzymes (Cofactors)-

-molecules/atoms/ions that assist in catalytic reactions

ex. Metal ions and Vitamins

II. Enzyme Kinetics -the study of the rates of an enzyme-catalyzed reaction

Topics:

1. Determination of maximum reaction velocity (V_max)

2. Binding affinities for substrates and inhibitors

3. Enzyme structures and chemistry

4. Rate analysis as a function of reaction conditions (temp, conc, inhibitors,etc)

A. Chemical Kinetics

1. For elementary reactions ( A --> P) or (A --> I --> J --> P )

a. Velocity = rate of a reaction

v = d[P] or v = -d[A]

dt dt

b. Rate law - mathematical relationship between rate and concentration of reactants:

v = k [A]

where k is the rate constant

for A --> P; where A is a single reactant, the molecularity = 1

(molecularity is the # of molecules that must simultaneously react)

Therefore: v is considered first order to [A].

k = v/[A] = (M/s) /M = s^-1

c. Order- given by the exponent of a reactant in a rate equation

2. For bimolecular reactions A + B --> P + Q

rate law: v = k [A] [B] reaction is 1^st order for A or B

but 2^nd order for A and B

k = v / ([A] [B]) = (M/s) / ( M * M) = M^-1 s^-1

2^nd order reactions have units of (M^-1 s^-1)

B. Free Energy of Activation

1. transition state- the intermediate state between reactant and product

-defined where the apex exists on an energy diagram

-the rate of a reaction is proportional to the concentration of reactant that can reach the transition state.

2. Free energy of Activation- energy differences between reactant and transition state. Gif

Arrhenius Equation:

So... k is inversely proportional to Ea, therefore if we can lower activation energy the rate should increase.

Question: How do we reduce activation energy?

a. Increase temperature of reactants

- an increase of 10^oC, will effectively double the reaction rate

b. Introduce a catalyst (enzyme)

-these cannot be consumed in the reaction

-do not affect the free energy of the reaction

C. Enzyme-catalyzed Reaction Kinetics

Tutorial: Writing rate law for enzymatically catalyzed processes

Recap: v a [A]; as [A] increases, then v increases

So what about enzyme kinetics, is v a [A]?

Answer: Yes and No

1. At low [A]: v a [A]

-this demonstrates 1^st order with respect to [A]

2. at higher [A]: v not a [A]

- this demonstrates 0 order with respect to [A]

a. Saturation Effect- when v doesn't change as a function of [A] because the enzyme is saturated with substrate.

b. Substrate Saturation Curve- rectangular hyperbola

V_max = the maximum limit for v. Asymptotic definition

Animation: Michaelis-Menten graph & enzyme kinetics

D. Michaelis-Menten Equation -model that relates velocity to reactant concentrations

1. Assumption: an enzyme & substrate associate reversibly to form an enzyme-substrate complex

v_f = k₁ [E] [S] and v_r = k_-1 [ES]

Image: Enzyme-substrate complex

** If the reaction is at equilibrium, v_f and v_r are equal, therefore:

k₁ [E] [S] = k_-1 [ES] and,

[E] [S] = ^k-1 = K_s (enzyme:substrate dissociation constant)

[ES] k₁

2^nd Step: Kinetics of Product formation

Image: Steady state assumption

2. Michealis Constant- relates the 3 rates associated with product formation

: importance K_m = [S] when v = ¹/₂ V_max

3. Michaelis-Menten Equation

Deriving: Michaelis-Menten Equation

a. Constraints on Michaelis-Menten

1. The reaction involves only 1 substrate

2. The reaction ES --> E + P is irreversible

3. [S]₀ > [E_T] and [E_T] is held constant

4. All experimental variables are held constant

Does the graph of v vs. [S] tell us much?

-the shape of the graph is a rectangular hyperbola and therefore Vmax is defined as the asymptotic function

of the graph, derived from experimental data where [S] approaches infinity. Km is derived from this.

So, the answer is NO.

Tutorial. Michaelis-Menten graphs

So why bother with v vs. [S]?

-Well I am glad you asked. We have better ways of representing this information

4. Lineweaver-Burk Double-Reciprocal Graph is the reciprocate of Michaelis-Menten

Does Lineweaver-Burk look similar to a linear equation?

III. Enzyme Inhibition

All enzymes have the ability to be regulated/inhibited.

-What this means is the kinetics (Km and Vmax) will be modified somehow.

A. What is an inhibitor

Enzyme inhibitors are molecules/ions that bind either to the active site of the enzyme or some adjacent site

but function to decrease the activity of the enzyme.

1. examples

a. Penicillin- binds with an enzyme glycoprotein peptidase. An enzyme that functions in creating the cell

walls of bacteria (halts peptidoglycan production)

b. Aspirin- binds with prostaglandin endoperoxide synthase. Prostaglandins are fat derivatives that function

in the inflammation response.

c. Ibuprofen- binds with cyclooxygenase- An enzyme that produces Progstaglandin precursors from

Arachidonic Acid.

B. Types of Inhibitors

1. Reversible- interact with the enzyme through noncovalent association/dissociation reactions.

a. Competitive- where the substrate and inhibitor compete for the active site.

E + I --> EI and/or E + I + S --> IES

- high [S] can overcome the effect of I.

What does this mean?

1. At a given [I], v decreases or 1/v increases

2. V_max does not be change

3. K_m increases (Apparent K_m or K_mapp)

Example: Malonate and Succinate Dehydrogenase : Malonate is an inhibitor of cellular respiration, because it binds to the active site of the succinate dehydrogenase in the citric acid cycle but does not react, thereby competing with succinate. For the oxidative phosphorylation reaction, Malonate is an inhibitor for complex II which, again, contains succinate dehydrogenase

b. Noncompetitive Inhibitors- bind with enzyme at a site other than the active site.

-a high [S] does not overcome the effect of I.

1. Pure noncompetitive- does not affect the binding of the substrate

K_I K_I'

E + I --> EI and ES + I --> IES