Biochemistry Kaplan Chapter 2 MCAT

Enzymes

Biological catalysts

1) lower the activation energy

--- make it easier for the substrate to reach the transition state

2) increase the rate of the reaction

--- can affect how quickly a reaction gets to equilibrium but not the actual equilibrium state itself

--- many important reactions can occur in a reasonable amount of time

**one enzyme can act on many, many molecules of substrate over time because it is not used up by the reactionAc**

Catalysts

Do not impact the thermodynamics of a biological reaction (deltaH and equilibrium do not change)

-Make reaction proceed at a much faster rate

Enzyme Specificity

A given enzyme will only catalyze a single reaction or class of reactions with a substrate

Oxioreductases

Catalyze oxidation-reduction reactions

-the transfer of electrons b/t biological molecules

Often have a cofactor that acts as an electron carrier, such as NAD+ or NADP+

Reductant = electron donor

Oxidant = electron acceptor

--enzymes w/ dehydrogenase or reductase in the names are usually oxidoreductases

Transferases

Catalyze the movement of a functional group from one molecule to another

Ex.) Kinases - Catalyze the transfer of a phosphate group, generally from ATP, to another molecule

Hydrolases

Catalyze the breaking of a compound into 2 molecules using the addition of water

Ex.) Phosphatases - cleaves a phosphate group from another molecule

i.e. peptidases, nucleases, and lipases

Lyases

Catalyze the cleavage of a single molecule into 2 products:

--do not require water

--do not act as oxidoreductases

*ALSO* catalyzes the synthesis of 2 molecules into a single molecule

Ex.) synthases

Isomerases

Catalyze the rearrangement of bonds w/in a molecule

(between stereoisomers & constitutional isomers)

==> some can be classified as oxidoreductases, transferases, or lyases depending on mechanism

Ligases

Catalyze addition or synthesis reactions, generally b/t large similar molecules, and often require ATP

==> most likely to be encountered in nucleic acid synthesis and repair

Ex.) DNA ligase puts DNA strands back together

Endergonic Reaction

A reaction that requires energy input (delta G > 0)

Exergonic Reaction

A reaction in which energy is given off (delta G < 0)

Active Site

-The location w/in the enzyme where the substrate is held during the chemical reaction

-Assumes a defined spatial arrangement in the enzyme-substrate complex that dictates the specificity of that enzyme for a molecule or group of molecules

-----Hydrogen bonding, ionic interactions, and transient covalent bonds w/in the active site all stabilize this spatial arrangement and contribute to the efficiency of the enzyme

Lock and Key Theory

Suggests that the enzyme's active site (lock) is already in the appropriate conformation for the substrate (key) to bind

--No alteration of the tertiary or quaternary structure is necessary upon binding of the substrate

Induced Fit Model

*More scientifically accepted theory*

The shape of the active site becomes truly complementary only after the substrate begins binding to the enzyme

The substrate has induced a change in he shape of the enzyme -> requires energy = endergonic

The substrate detaches from the enzyme and enzyme returns to original shape -> no energy = exergonic

Cofactors and Coenzymes

-non protein, small molecules that can bind to the active site of an enzyme and participate in catalyzing the reaction (use ionization, protonation, deprotonation)

-cofactors: usually inorganic molecules or metal ions

-coenzymes: small organic groups such as vitamins or NAD, FAD, CoA

Apoenzymes

Enzymes without their cofactors

Holoenzymes

Enzymes with their cofactors

Prosthetic Groups

Tightly bound cofactors or coenzymes that are necessary for enzyme function

Kinetics of Monomeric Enzymes

The concentrations of the substrate [S] and enzyme [E] greatly affect how quickly a reaction will occur

- E > S ; there are many active sites available, quickly forms products and reaches equilibrium fast

- S increases ; the rate of the reaction increases

- S ~ E ; as S gets closer to E the rate of reaction levels off and reaches a maximal rate , until all active sites are occupied

--- Adding more S will not change the rate of the reaction = Saturation

Reaction Velocity : y = (x)^1/2

[E] = [S] at Vmax

Km = [S] at 1/2 Vmax

Michaelis-Menten Equation

For most enzymes, describes how the rate of the reaction (v) depends on the concentration of both E and S which forms the product [P].

Enzyme-Substrate Complexes:

-- form at a rate K1

E + S -> E + P

-- can either dissociate at a rate K-1

OR

-- turn into E + P at a rate Kcat

Concentration of the enzyme will be kept constant

v = (Vmax [S]) / (Km + [S])

When the reaction rate is equal to half of Vmax, Km = [S]

**Km is the substrate concentration at which half of the enzyme's active sites are full**

Michaelis Constant

Km - used to compare enzymes - it is an intrinsic property of the enzyme-substrate system and cannot be altered by changing the concentration of substrate or enzyme

- Higher the Km = Lower affinity for substrate

- Lower the Km = Higher affinity for substrate

--- Low [S] required for 50% enzyme saturation

Vmax

Represents maximum enzyme velocity and is measured in moles of enzyme per second

Vmax = [E]kcat

OR

v = (kcat [E][S]) / (Km + [S])

kcat -> measures the # of substrate molecules converted to product per enzyme molecule per second

Most enzymes have kcat values b/t 101 and 103

Catalytic Efficiency

The ratio of kcat / Km

- A large kcat (high turnover) or small Km (high substrate affinity) will result in a higher catalytic efficiency = more efficient enzyme

Lineweaver-Burk Plots

Useful when determining the type of inhibition that an enzyme is experiencing b/c vmax and Km can be compared w/o estimation

X intercept = -1/Km

y intercept = 1/vmax

Cooperativity

-Enzymes that have multiple subunits and active sites

-Subunits & enzymes may exist in 2 states:

--- 1) low-affinity tense state (T)

--- 2) high-affinity relaxed state (R)

**Binding of the substrate encourages the transition of other subunits from the T state to the R state, increasing the likelihood of substrate binding by other subunits**

**results in characteristic sigmoidal curve on a Michaelis-Menten plot**

Ex.) hemoglobin, regulatory enzymes i.e. Phosphofructokinase-1 (PFK-1)

Hill's Coefficient

greater than 1 = has positive cooperativity

1 = no exhibit of cooperative binding

less than 1 = has negative cooperativity

the higher it is, the more cooperative it is.

Temperature and Enzymes

Enzyme-catalyzed reactions tend to double in velocity for every 10 degrees Celsius increase in temp. until the optimum temp. is reached

-- in the human body this is 37 degrees Celsius

-- beyond this activity falls off sharply as the enzymes denature at high temp.

pH and Enzymes

pH affects the ionization of the active site and can lead to denaturation of the enzyme

-- in human blood the optimal pH is 7.4

-- a pH lower than 7.35 in human blood is deemed acidemia b/c it is more acidic than physiologically neutral 7.4

**Exceptions: pepsin in the stomach has maximal activity at pH of 2 and pancreatic enzymes of the small intestine work best at pH of 8.5**

Salinity and Enzymes

Increasing levels of salt can disrupt hydrogen and ionic bonds, causing partial change in conformation of the enzyme and causing denaturation

Negative Feedback (feedback inhibition)

Helps maintain homeostasis: once there is enough of a given product the pathway that creates the product is turned off

-- Product may bind to the active site of an enzyme , thus competitively inhibiting these enzymes and making them unavailable

Competitive Inhibition

Occupancy of the active site

- Substrates cannot access enzymatic binding sites if there is an inhibitor in the way

- *Does NOT alter the value of vmax*

- *DOES increase the measured value of Km*

--- b/c substrate concentration has to be higher to reach 1/2 vmax in the presence of inhibitor

- *Can be overcome by adding more substrate so that the substrate-to-inhibitor ratio is higher*

--- enzyme will be more likely to bind substrate than inhibitor => assuming the enzyme has equal affinity for both

Noncompetitive Inhibition

Binds to an allosteric site instead of the active site which induces a change in enzyme conformation.

**Decreases the measured value of vmax b/c there is less enzyme available to react**

**Km is unchanged*

-- the 2 molecules do not compete for the same site, thus inhibition CANNOT be overcome by adding more substrate

-- binds equally well to the enzyme-substrate complex

Mixed Inhibition

Inhibitor can bind to either the enzyme or the enzyme-substrate complex but has different affinity for each

-- if it had the same affinity for both it would be a noncompetitive inhibitor

-- *vmax is decreased*

Bind at an allosteric site

*If inhibitor preferentially binds to the enzyme, it increases the Km value (lowers affinity) and if it binds to the enzyme-substrate complex it decreases the Km value (increased affinity)*

Uncompetitive Inhibition

Inhibitors bind only to the enzyme-substrate complex and essentially lock the substrate in the enzyme, preventing its release

-- *increasing affinity b/t the enzyme and substrate*

-Allosteric Site

-Lowers Km value

-Lowers vmax

Irreversible Inhibition

active site is made unavailable for prolonged period of time or enzyme is permanently altered

Allosteric Enzymes

Have multiple binding sites, and alternate between an active and an inactive form. Those that bind to the allosteric sites are either activators or inhibitors.

Covalently Modified Enzymes

enzymes can be activated or deactivated by phosphorylation or dephosphorylation

- one cannot predict whether these will activate an enzyme w/o experimental determination

Can also be modified by covalent attachment of sugar groups (glycosylation)

- can tag an enzyme for transport, or modify protein activity and selectivity

Zymogens

Contain a catalytic (active) domain and regulatory domain; regulatory domain must be either removed or altered to expose the active site

- It is critical that certain enzymes (like the digestive enzymes of the pancreas) remain inactive until arriving at their target site; too dangerous

* Most have the suffix -ogen *

Ex.) trysinogen