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1

Define the characteristics of enzymes

1. Accelerate reaction rates but do NOT change the equilibrium;
2. Decrease the activation energy of the reaction (stabilize the transition state)
3. proteins or ribozymes (RNA molecules)
4. 25% of human genes are coded for enzymes
5. Highly specific in reactions they perform and in their substrates

2

What is the difference between the kinetic and thermodynamic parameters of chemical reactions?

For a given reversible reaction:

Kinetic: Activation Energy (energy from the reactant ground state and the transition state) determines the reaction rate; says nothing about the preferred direction. Note, Ea is the barrier in EITHER direction

Thermodynamic: only one direction releases free energy (G) and is the spontaneous reaction, and this occurs only if the difference in free energy between the reactant and the product is a negative value; SPONTANEITY IS INDEPENDENT OF THE PATH TAKEN TO COMPLETE THE TRANSITION IN THE RXN.

3

What is the transition state?

the activated species with the highest energy (T*)

4

What happens when the energy of the reactants is greater than the energy of the products?

the delta G is negative and so the reaction releases energy (exergonic) and spontaneous.. opposite for when the delta G is positive

5

What effect does an enzyme have on the kinetic and thermodynamic barriers?

Kinetic: enzyme decreases the activation energy, which in turn increases the reaction rate IN BOTH DIRECTIONS because the transition state is at a lowered energy level; speeds up the reaction (For each direction)

SINCE THE REACTION RATE IS INCREASED IN BOTH DIRECTIONS, the thermodynamic barrier is the same for the catalyzed and uncatalyzed reaction (Keq DOES NOT CHANGE)

6

Why is the MIchaelis Menten reaction plot of initial velocity vs. [S] for an enzyme hyperbolic and not linear?

Linear: without an enzyme the velocity (speed of reaction) increases linearly with the reactant concentration

Hyperbolic: in enzymes the hyperbola indicates saturation kinetics; at a fixed enzyme concentration a non-linear relationship between velocity and reactant concentration; because at a certain point the rate of the reaction is independent of the concentration of the [S] (at Vmax). This is because the rate of the reaction is limited by the intrinsic properties of the enzyme (its turnover rate essentially)

** NOTE: at VERY low [S] relative to the Km the velocity increases LINEARLY with [S] indicating maximal dependence; at VERY high [S] >>>> Km the velocity approaches the point that is completely independent of [S] (occurs at Vmax)

7

What are the three assumptions of the Michaelis Menten equation?

1. that substrate concentration is in molar excess over the enzyme concentration
2. The initial velocity of the reaction is measured (thus k4 does not exist)
3. That at steady state the rate of the enzyme substrate complex (ES) is equal to the rate of its dissociation (either into E + P or E + S)

8

Describe all of the terms in the Michaelis Menten equation

v = Vmax [S] / Km + [S];

velocity is proportional to the [ES] and if the enzyme is 100% saturated then [ES] = [E]t so the velocity is really proprtional to the total enzyme concentration

** reaction velocity indicates how much product is made in a define time period (mg per time or moles per time)

Vmax = maximal velocity = the fastes the enzyme catalyzed reaction can go

Km = the concentration of substrate and half max velocity

9

How is the velocity of the enzyme reaction described at vmax?

Vmax = kcat [E]total

where k cat = the reaction rate for ES --> E + P (first order rate constant)

10

what is the difference between turnover and specific activity? How are they each measured?

Turnover: the amount of product produced by an enzyme in a given amount of time: (how efficiently does the enzyme turn over the substrate into product); measure as sec -1 ( kcat)

Specific Activity: a measure of how "pure" a solution is; it tells you how much of the total protein present is the enzyme of interest at that specific time = SA; measure in units per mg protein (units = 1 umole/min); so its measured in umoles enzyme/ mg total protein * min


1 Unit = umole/ min
if you know the molar [enzyme] then you can calculate Kcat (turnover) = time -1
if you don't know the molar [enzyme] but you can measure the total amount of protein, then you can calculate specific activity = U/mg of protein

11

What is Km in the michaelis menton enzyme equations?

Km = michaelis constant;
defines [ES] in the stead state (Km = [E] [S] /[ ES]
equals the substrate concentration that gives half maximal velocity thus if Km increases then more substrate is needed to achieve half maximal velocity (can be used to interpret affinity of an enzyme for substrate)

**** if Km = [S] then the reaction is at i/2 Vmax

12

what is the concentration of a metabolite / S typically in vivo?

In vivo the concentration of a given metabolite in cells is typically in the range of the Km value of the enzyme ([S] ~ = Km)

13

Describe the Lineweaver Burke plot and where all of the values of the M - M equation can be found

Y axis = 1/ V
X axis = 1/ [S]
Y intercept = 1/ Vmax
X intercept = -1/Km
slope = Km/ Vmax

14

Is Km a dissociation constant?


** even though Km = same equation at Kd (dissociation constant) with respect to concentration of the substrate, Km IS NOT THE DISSOCIATION CONSTANT unless k 3 <<<< k2 because then Kd = K2/ K1

Kd is defines by 2 rate constants (K2 /K2)
Km is defined by 3 rate constants (K2 + K3) / K1

15

what is the rate of an enzyme-catalyzed reaction dependent on? what is the relationship?

when the substrate is not limiting, the reaction rate is ALWAYS LINERARLY dependent on [E]

16

How is the rate of an enzyme -catalyzed reaction related to [S]?

the reaction rate increases hyperbolically with increases in [S] `

17

What are the 6 different classes of enzymes? describe each

1. Oxidoreductases: transfer reducing equivalents (H2) from one redox system to another (AH2 + B + E --> A + BH2 + E)
2. Transferases: transfer functional groups from one substrate to another (AC + B + E --> A + BC + E)
3. Hydrolases: catalyze hydrolysis reaction (the addition of water to (AB + H2O + E --> HA + BOH + E )
4.Lyses: catalyze the removal of groups of atoms without water (AB + E --> A + B + E)
5.Isomerases: catalyse isomerization reactions by rearranging atoms in the molecules (A + E --> Aiso + E)
6.Ligases : use ATP to catalyze the joining of two molecules (A+B+E + ATP --> AB + E + ADP + Pi)

18

How does a substrate interact with the enzyme ?

substrate binds to the active site of an enzyme via non-covalent interactions between the amino acids in the active site and the substate; interactions = H bonding, electrostatic, hydrophobc (active sites usually hydrophobic to prevent weakened interactions between ions due to waters high dielectric constant); NOTE: the amino acids involved in substrate binding and catalysis are NOT adjacent to each other in the linear protein

Two main methods of attachment
1. Lock and Key model: enzyme substrate are perfect shapes for each other and form a tight bond
2. Induced fit model: the enzyme and substrate are slightly off shape so either substrate or the active site needs to shift its shape so that they can fit.

also, some enzymes require a small molecules already located at the binding site (a cofactor) in order to bind the substrate

19

What are the main ways in which an enzymes lowers the energy of activation and decreases the energy of the transition state so the reaction goes faster?

1. Orientation and Proximity: E binds S and holds it in the correct orientation for catalysis; can be intermolecular or intramolecular
2. Substrate Strain: enzyme can impose strain or distortion on the substrate so that it binds the E
3. General Acid Base Catalysis: side chains of aa in the active site can act as H donor/acceptor and facilitate rxn
4. Metal ion catalysis: passive (ion involved in a stabilization of the coordination bond) or active (ion needed for the catalytic rxn)
5. Covalent Catalysis: In the reaction BX +Y --> BY X the side chain of enzyme aa better attacker than Y and better leaving group than X
6. Environmental Factors: an optimium for temperature and pH (proteins denature above a certain temperature and pH influences ionization of the E's aa side chains and of the S leading to variations in stability, biniding, and rate of mechanism

20

What is unique thing about lysozyme?

1. strain on substrate so one of the sugars is forced to assume half chair conformation
2. pKa of Glu-35 is higher than measured for free aa, so at pH 5 the Glu -35 is abnormally protonated

21

What is interesting about the serine proteases with respect tot their active sites/protein structure?

They are basically the same exact thing except for that the active site is different;
1. chymotrypsin Active site = large and nonpolar (A G)
2. Trypsin active site has an aspartic acid residue
3. Elastase : the active site is constricted

22

Describe the three types of reversible inhibition

1. Competitive Inhibitor: binds to substrate binding site so it competes with the substrate; causes a DECREASE IN KM but does not change Vmax; it can be overcome by an increase in [S]
2. Noncompetitive Inhibitor: binds at a site different from the binding site therefore it is INDEPENDENT OF THE SUBSTRATE (does not compete with it) causes a DECREASE IN VMAX no change in KM
3. Uncompetitive Inhibitor: binds to the enzyme AFTER the substrate has bound; causes the KM TO decrease and the VMAX TO DECREASE; can be overcome by an increase in [S]

23

How does a a decreased affinity related to Km?

increased Km

24

which form of reversible inhibition is most similar to the irreversible inhibition? how?

noncompetitive because both of them cause a decrease in Vmax

25

What is a biochemical cofactor? What is an inorganic biochemical cofactor (ex)? organic biochemical cofactor?

cofactor = NON PROTEINACIOUS chemical compound that is BOUND TO A PROTEIN and is required for the biolofical activity of the protein;
(helper molecules that assist in the biochemical transformations)
inorganic cofactor = metal ions (help balance out charges on molecules they associate with), transition ions (they can exist in 2+ oxidation state)
organic cofactors = vitamin derivatives ( ie: fat soluble or water soluble)

26

what is a coenzyme? a prosthetic group? apoprotein?

coenzyme = cofactor that is loosely bound
prosthetic group = cofactor that is tightly bound
apoprotein = an inactive protein without its cofactor

27

What is ATP's cofactor? why?

Mg 2+ because the 2+ is needed to balance out ATP's negative charge

28

Describe the structural components of vitamins

Fat soluble: have a long hydrophobic polyisoprene chain; include vit KADE
Water soluable: polar molecules

29

What is the principal function of Vitamin A? How does it form other forms ? retinal?

retinal = signal transduction
retinoic acid = hormone

Beta-Carotene is a pro-vitamin; it's center is cleaved to yield a group of molecules (retinoids) collectively known as vitamin A if the C terminal is an alcohol = retinol, acid = retinoic acid, aldehyde = retinal

Retinal is involved in signal transduction; its the cofactor for rhodopsin and when it absorbs a photon it changes to all trans which causes a change in opsin and triggers a G protein eventually leading to a nerve response.

30

What vitamins can act as hormones?

Vitamin A (retinoic acid)
Vitamin D