Enzymes Flashcards
Highly specific, extremely fast, biological catalysts
-Proteins
Enzymes
Mutations in proteins and enzymes are the cause of many
Diseases
Enzymes can be used as drugs, an example of this is the clot buster
Activase
Enzymatic reactions are multi-step reactions. The first step in an enzyme reaction is the enzyme binds
Substrate (forms the ES complex)
The second step in an enzyme reaction is the
conversion of ES to EP
The third step of an enzymatic reaction is the
Dissociation of P and regeneration of E
A molecule that accelerates a chemical reaction and is regenerated at the end of the reaction
Catalyst
Says that an enzyme has an active site that fits only a specific substrate
-ex: yeast fermented the D- but not L- forms of glucose, mannose, and galactose
Fisher’s lock and key model
A three dimensional cleft formed by catalytic amino acids that come from different parts of the protein sequence
Enzymes active site
Bind substrates but don’t carry out a chemical reaction
Receptors
Bind substrates and carry out chemical reactions
Enzymes
Close together in tertiary structure, but not in primary structure
Catalytic amino acids
Only a very small portion of an enzymes aminos function in the active site, the rest serve as a
Scaffold
Small pockets lined with a few catalytic amino acids that participate in substrate binding and catalysis
Active site
Small part of the total volume of the enzyme and is non-polar (excludes water unless water is a reactant)
Active site
The nonpolar characteristics of active sites enhances substrate binding by increasing
Electrostatic interactions
Substrates bind active sites by way of many
weak non-covalent interactions
What is the energy for a
- ) Covalent interaction?
- ) Non-covalent interaction
- ) 50 kcal/mol
2. ) 0.5-2 kcal/mol
Enzymes can undergo many catalytic cycles because substrates and products bind
reversibly
Precise active site conformation of glucokinase explains specific binding and reaction of ATP with glucose but not galactose. Why does galactose not bind even though the two differ by only a single hydroxyl?
Cooperative binding
The lock and key model proved to be inadequate when looking at glucokinase and hexokinase because
Since they bound glucose, they should have been able to bind water and react with ATP, but they could not
The lock and key model failed to explain why ATP did not react with water, which paved the way for the
Induced-fit model
Summarize the induced fit model
Substrate binding causes a conformational shift in the enzyme, which stabilizes the active conformation and allows catalysis
In the induced fit model, a specific substrate activates the enzyme by
Orienting catalytic groups on the enzyme
What was the experimental evidence supporting the induced fit model?
The binding of glucose induced a large conformational change in glucokinase
In the case of ATP transferring a phosphate to NMP rather than water, we see that the induced fit conformational change assures that a catalytically competent complex is formed only when
-prevents the reaction with water
Both ATP and NMP are bound to NMP kinase
Enzymes increase the rates of reactions by a factor of
10^6-10^17 times
The minimum energy required for two molecules to react
-inversely proportional to reaction rate
Activation energy
Enzymes work by decreasing the
Activation barrier (transition state energy)
Enzymes have no effect on the
Gibbs free energy
Enzymes create a new reaction pathway with a lower activation energy through specific binding to the
Transition state
How do enzymes lower the transition state energy?
Tighter binding to transition state than to substrate
What are five ways enzymes speed up the rate of a reaction?
- ) Proximity (all)
- ) Stabilization of transition state (all)
- ) Covalent or nucleophilic catalysis (some)
- ) Acid-base catalysis (most)
- ) metal ion catalysis (many)
Increase the effective concentration of reactants that are in the proper orientation
Proximity
In a chain of successive reactions, the rate enhancement due to proximity increases as the
Products structure becomes more rigid (less ability to be in improper orientation)
What are the three enzymatic strategies employed by the protease enzyme chymotrypsin?
- ) Nucleophilic or covalent catalysis
- ) General acid-base catalysis
- ) Transition state stabilization (oxyanion hole)
The serine residue in the active sight of chymotrypsin is a powerful
Nucleophile (but must be deprotonated)
The pKa of the side chain of serine is
pKa = 13
The serine residue in the active site of chymotrypsin is turned into a potent nucleophile by deprotination using
Acid-base catalysis
The extremely reactive serine residue in chymotrypsin is created by the
Catalytic triad (Ser, His, Asp)
-His accepts a proton to become HisH+
In the chymotrypsin active site, once serine is deprotonated, it
Nucleophilically attacks the carbonyl carbon of amino acid, forming the tetrahedral intermediate (oxyanion hole), which then reforms the double bond and kicks off the amine
Serine nucleophilically attacking the carbonyl carbon is an example of
Covalent Catalysis
Binds the transition state tightly and stabilizes the transition state by way of H-bonds which were not possible in the reactant
Oxyanion hole
A space in the enzyme active site that is ready to bind a negatively charged group
Oxyanion hole
Summarize how chymotrypsin cleaves at the C-terminal end of the aromatic amino acids (Phe, Tyr. Trp)
Aspartate H-bonds w/ histidine, the His Nitrogen attacks the serine OH,allowing the SerO- to nucleophilically attack the carbonyl carbon of the amino, forming the tetrahedral intermediate. The oxyanion hole stabilizes the intermediate until the carbonyl is reformed, breaking the amide bond and the amine fragment is released. His then takes a proton from water, which generates OH, which attacks the carbonyl carbon of the residue still bound to serine, forming the tetrahedral intermidiate. The electrons reform the double bond, releasing a now carboxylic acid from serine. Serine takes a proton back from histidine.
A reaction where ΔG
Exergonic Reaction
A reaction where ΔG > 0
-Non spontaneous reaction
Endergonic reaction
An endergonic reaction can be driven forward by
Coupling it to an exergonic reaction
What is the ΔG˚ of ATP hydrolysis?
-30.5 kJ/mol
What are three reasons that ATP hydrolysis is so favorable?
- ) ADP and Pi (products) are more stable than ATP (reactant)
- ) Electrostatic repulsion (ADP has -2 charge and ATP has -3)
- ) Resonance stabilization of Pi
What is a great example of a coupling reaction?
Coupling the phosphorylation of Glucose to form glucose-6-phosphate with ATP hydrolysis
The ΔG˚ value provides absolutely no information about
Reaction rates
Kcat is the rate constant of
ES —> E + P
The velocity of an enzyme reaction is proportional to the
Concentration of the ES complex i.e. [ES]
How can we use the MM equation to determine Kcat and Km?
Measure velocity of the reaction
How do we measure the velocity (speed) of the reaction?
Measure the change in concentration of substrate or product over the time of the enzymatic reaction
The MM equation shows that enzymatic rate/ velocity increases linearly with
Enzyme concentration
The MM equation shows that enzymatic rate increases asymptomatically with
Increasing [S]
On a plot of velocity vs [S], Km can be thought of as
Vmax/2
On a plot of velocity vs [S], Kcat can be thought of as
Vmax/[E]
The ratio of Kcat/Km is called the
-measure of enzyme efficiency
Specificity constant
From a definition standpoint, we can think of Km as being inversely proportional to the
The enzymes affinity for substrate
- Large Km = small affinity
- Small Km = Large affinity
Result in a high specificity constant
- ) Rapid Turnover (large kcat)
2. ) High enzyme affinity for substrate (Low Km)
A measure of the affinity of the substrate for the enzyme
Km
Characteristic for an enzyme for a particular substrate
-Changes with pH or Temperature
Km
Km values are usually close to
Physiological substrate concentration
The maximum velocity with which the enzyme can catalyze the reaction
Vmax (units = M/s)
The turnover number of an enzyme, representing the number of moles of substrate converted to products persecond per mol of enzyme
Kcat (units s^-1)
Vmax is linearly dependent on
[E]
Some enzymes require cofactors. An enzyme and it’s cofactor are called a
Holoenzyme
Small organic molecules, mostly derived from vitamins, that are either noncovalently bound to the enzyme, or covalently bound (prosthetic groups)
Coenzymes
Apoenzymes without their cofactor are
Inactive
Provide the functionalities not found in the natural amino acids
Coenzymes
Have different primary structures or amino acid sequence, but catalyze the same chemical reaction and act upon the same substrate
-Thought to have evolved from gene duplication and divergence
Isozymes
Allow fine tuning of metabolism to meet the needs of a given tissue or developing stage
Isozymes
Do isozymes have the same Km and kcat as their enzyme isomers?
No
Measurement of isozyme levels helps in
diagnosis
What happens within one day of heart muscle damage?
-indicates myocardial infarction
- ) LDH isozyme H4 increases within 12-24 hrs.
2. ) Level of MB creatine kinase in the blood increases
Enzyme activity is affected by temperature. Explain how temperature increases rate.
As temp increases, there are more molecular collisions between E and S
Enzyme activity is affected by temperature. Explain how temperature decreases rate.
When temp gets too high, proteins are denatured, causing the decrease in rate
What are the two negative effects that pH can have on an enzyme?
- ) extreme pH’s irreversibly denature the enzyme and activity is lost forever
- ) Can reversibly affect the enzyme by ionizing the functional groups involved in catalysis
Enzymes in different organs have different pH optimum, but most intracellular enzymes have a pH optimum of
pH = 7.2
Enzyme concentration is regulated by
- ) Synthesis (transcription, translation, etc.)
2. ) degradation (proteolysis)
Enzyme activity is regulated by compartmentalization in
Cell organelles
Enzyme activity is regulated by post translational modifications such as
Phosphorylation, glycosylation, methylation, etc.
Enzyme activity is regulated by regulatory proteins such as
Transcription Factors
Enzyme activity is inhibited by
Feedback inhibitors, enzyme inhibitors, and products
ADP-ribosylation of EF2 blocks its capacity to carry out translocation of the growing polypeptide chain, thus
-Accounts for remarkable toxicity of diptheria toxin
Protein synthesis ceases
Over 95% of protein phosphorylation occurs on
Serine resiues
Highly negatively charged phosphoryl group can alter
Substrate binding and catalytic activity
Is reversible and fast and it can either activate or inhibit the enzyme
Phosphorylation
Abnormal levels of protein phosphorylation are a cause or consequence of major diseases such as
Cancer, diabetis, and arthritis
What is an irreversible way to activate enzymes?
Proteolysis
-apoptosis mediated by caspases from procaspases
A prime example of feedback regulation of an enzyme is that of the allosteric enzyme Aspartate Transcarbamylase, which is activated by? inhibited by?
Activated by ATP
Inhibited by CTP
Enzymes that change their conformational ensemble upon binding of an effector, which results in an apparent change in binding affinity at a different ligand binding site.
Allosteric Enzymes
All allosteric enzymes are
Oligomeric
Allosteric molecules (bind in the allosteric site) do not resemble the
Substrate
Allosteric enzymes can be distinguished kinetically because they show
Sigmoidal curves
Do allosteric enzymes follow MM kinetics?
No
Enzymes following MM kinetics show a
Hyperbolic curve
Allosteric curves are sigmoidal due to the allosteric enzyme’s
Cooperative binding
Allosteric enzymes exist in which two equilibrium conformations?
- ) Active (R) state
2. ) Inactive (T) state
Binds substrate better and/or has higher catalytic activity
R-state
How does an allosteric activator increase substrate binding and/or activity?
Binds to the allosteric site and stabilizes the R-state
How does an allosteric inhibitor decrease substrate binding and/or activity?
Binds to the allosteric site and stabilizes the inactive T-state
In AcT, the inactive T-state is favored by
CTP binding
Inhibitors shift the allosteric sigmoidal curve to the?
Right
Activators shift the allosteric sigmoidal curve to the?
Left
The basis for clinical drug therapies: antiviral, antibacterial, and antitumor therapies
Enzyme inhibition by small molecules
What are the two main classes of inhibitors?
- ) Irreversible inhibitors (bind covalently)
2. ) Reversible inhibitor (bind reversibly)
What are the three types of reversible inhibitors?
- ) Competitive
- ) Noncompetitive
- ) Uncompetitive
Substrate analogs that form a covalent bond with and active site amino acid
-ex: penicillin bonding to transpeptidase
Irreversible inhibitors
Aspirin is an example of an
Irreversible inhibitor
Bind the same active site as the substrate
Competitive inhibitor
Bind to a site separate from the active site
Noncompetitive inhibitor
Stable molecules that resemble geometric and/or electronic features of the highly unstable transition state.
-Potent inhibitors
Transition state analogues
What are the kinetic features of a competitive inhibitor?
- Vmax is unchanged
- Km increases
No inhibition at high [S]
What are the kinetic features of a noncompetitive inhibitor?
- Km is unchanged
- Vmax decreases
-High [S] does not overcome inhibition
Where do the lines intersect on a linweaver-burke plot for
- ) competitive inhibition?
- ) noncompetitive inhibition?
- ) y-axis
2. ) negative x-axis
The most widely prescribed drug that lowers cholesterol
Statins (competitive inhibition)
Statins act by inhibiting
HMG-CoA reductase
What is Pi?
Inorganic Phosphate
The gibbs free energy represents the
Maximal work under a set of conditions
ATP is so reactive because it has three phosphates and a charge of
-3 or -4, averaging to -3.5
Where do coupled reactions occur in the enzyme?
On the same active site
The dissociation constant of [ES]
Kcat
What are the trends of the [ES] dissociation constant Km?
The smaller the Km, the faster the reaction typically is
-Small Km means enzyme does not dissociate from [ES] and reaction proceeds
What is the rate limiting step of an enzymatic reaction?
Kcat
When [S] is in excess, the velocity increases linearly with
[E]
Why does the rate increase asymptomatically with increasing substrate concentration?
At some point all of the enzyme active sites are saturated with substrate, and Vmax has been reached
The higher the value of Km, the
slower the reaction
Mutations in enzymes that affect the ability of substrate to bind will also effect
Km
What are the units of Km?
M, mM, or uM
Is roughly equal to the physiological substrate concentration
Km
What is the fastest known enzyme?
-Called the perfect enzyme
Carbonic anhydrase
An inactive enzyme without its cofactor
Apoenzyme
Allow variability that expands beyond that of the 20 amino acids in enzymes
Coenzymes
What is a common example of isozymes?
Hexokinase and Glucokinase
The liver synthesizes and utilizes
Glucokinase
The optimum temperature of human enzymes is
38 degrees celcius
Km and Kcat values are affected by
Temperature and pH
Why is pH so important to enzymes?
The H-bonds that form from the catalytic aminos will not be possible if pH is outside of optimum range
We don’t want to have to synthesize an enzyme every time we need it, because that will take to long. So, we synthesize enzymes and keep them in the inactive form called a
Zymogen
The activation of a zymogen to an enzyme is
Irreversible
Can be used to turn an enzyme off by inactivating a Ser, Thr, or Tyr in the active site
Phosphorylation
An example of enzyme regulation by specific proteolysis
Zymogen activation
Regulation of an enzyme at a distance (how many metabolic pathways are regulated)
Allosteric Regulation
All allosteric enzymes are oligomeric, meaning they are
Dimers, trimers, tetramers, etc
Do not look like the enzymes substrate, thus they do not bind to the active site. Instead, they bind to a regulatory site and cause some form of conformation change in the enzyme which either activates or inhibits the enzyme
Allosteric Effectors
Their association with an enzyme is not allosteric because they actually bind to the active site
Cofactors
Instead of a Km, allosteric enzymes have a
K0.5
What type of allosteric inhibitor or activator affects K0.5 but not Vmax?
K-type
What type of allosteric inhibitor or activator affects Vmax but not K0.5?
V-type
AcT is a hexamer, the active site is at the interface of two subunits in the T-state. When an activator is added, the interface is broken, freeing the active sites and stabilizing the
R states
Most drugs are
Inhibitors that target enzymes
