Download Catalytic Strategies I

FUNDAMENTALS 10:00-11:00
MONDAY, AUGUST 23rd, 2010
DR. LARRY DELUCAS
I.
ENZYME CATALYTIC STRATEGIES
Scribe: BO BRADFORD
Proof: ROSS ISBELL
Page 1 of 5
What are the magnitudes of enzyme induced rate acceleration [S2]
a. Enzymes dramatically accelerate biological reactions as compared to laboratory experiments ran in test tubes
b. Steric reasons why this happens, charge reasons why this happens, it involves water and desolvation.
c. Enzymes catalyze a rxn. 10/1000x faster than the chemists can do in a test tube- they are unable to
reproduce the combining of molecules. The enzyme & complicated nature of proteins and how they react to
closely related reactants provide ways to adapt to a charge and hold specific charges so that they can be
transferred.
d. Millions of reactions take place every second and they are all regulated precisely.
e. Accelerations of rxn.’s can be greater than 10x15, they all correspond to LARGE changes in free energy
f. Enzymes help to lower our free energy barrier in order to get to our ultimate product-this is why the enzyme is
favored over the substrate just reacting with itself
g. These reactions pass through a phase known as a transition state on their way to the product which is
different from than just the enzyme & substrate binding
i. There is a change in how the enzyme sits around the substrate
ii. T.s. is more stable than substrate binding, yet not too stable. It must have the capability to dissociate back
into the ultimate product.
iii. ALL BIOLOGICAL INTERACTIONS ARE WEAK. The same is true with enzymes; we want to create a
stable transition state complex, but not too stable because we still want to have the ability to form a
product.
h. Linus Pauling was one of the key people to study/delineate the transition state & study how it lowers the
activation energy as a compound interacts with an enzyme to create a final product.
II. What are the magnitudes of enzyme- induced rate accelerations [S3]
a. Compares the rates of reactions one could perform without enzymes in a test tube with biological reaction
rates using the enzymatic approach
III. What role does Transition State stabilization play in enzyme catalysis [S4]
a. There is a free energy to create a product known as DELTA-G.
b. You have substrates & you go up to transition states that transitions into the enzyme alone again & the
product
c. This barrier is quite high- an enzyme can lower the barrier.
d. When the substrate binds to the enzyme complex, there is a dip in free energy. If this dip is very favorable-it
comes down very low-the barrier to get to transition state and finally product is even higher & its unlikely that
you’ll have a great acceleration in creating the product
e. These graphs demonstrate how/when we form a transition state
f. If you look at enzymes & their speed there’s a correlation. [E][S]/[ES]= dissociation constant. (do the same
thing with the transition state). IN ORDER FOR THE ENZYME TO BE HELPFUL THE DISS. CONSTANT
FOR THE TRANSITION STATE MUST BE LESS THAN THE SUBSTRATE. You have to have an advantage
with the transition state. The enzyme must stabilize the transition state more than it stabilizes the ES.
g. Kt must be smaller than Ks.-the greater the difference of the ratio determines the overall enhancement of
reaction rate.
IV. How does destabilization of ES affect enzyme catalysis [S5]
a. How do we make transition state favorable? We don’t do anything…it is just that in an aqueous solution we
have substrate that is free & substrate that is bound to an enzyme. If the substrate has charges on it, or if it’s
polar it’s going to interact with its environment- its going to bind to the enzyme and to some extent desolvate
the enzyme. Basically, it goes from an ENTROPY HAPPY environment to an ENTROPY UNHAPPY. When
this occurs we lose our favorable entropy situation-this is one way to destabilize the ES complex, not causing
a low dip in the graph.
b. If you have an enzyme with a substrate that’s going to bind…the a.a. arrangement on the substrate is not
going to be conformationally perfect when it first comes into the enzyme. It is good enough that it can bind,
but when it does there can be steric strain (causing some distortion of the enzyme itself), charges that cause
unfavorable situations, you must rid water of the binding site-desolvation process. ALL of these three factors
make the ES complex not so favorable. We don’t want the barrier to be so large that we can’t ultimately get to
the transition state-the next step- easily.
c. Refers to book reading for more info…
V. How does destabilization of ES affect enzyme catalysis [S6]
a. Enzyme and substrate are brought together by a certain degree of free energy for favorable interactions b/t
the two
FUNDAMENTALS 10:00-11:00
Scribe: BO BRADFORD
MONDAY, AUGUST 23rd, 2010
Proof: ROSS ISBELL
DR. LARRY DELUCAS
ENZYME CATALYTIC STRATEGIES
Page 2 of 5
b. You have a.a. situated on the enzyme that interact favorably with the substrate. This is why they come
together, water leaves & substrate binds to enzyme.
c. Intrinsic binding energy is compensated by the entropy loss due to binding of substrate & enzyme as well as
steric strain, distortion, and desolvation. The culmination of all these factors, cause a smaller gain in free
energy- a smaller dip from the E + S to the ES.
d. Substrate wants to be with enzyme but not too much
e. Entropy is negative-the complex is in less favorable conformation, the overall free energy goes up. This is
why were higher (than the green dotted line)…strain, distortion, desolvation also contribute to this gain in free
energy.
VI. How does destabilization of ES affect enzyme catalysis [S7]
a. Without stabilization, you create a huge hill of free energy to climb in order to get to the transition state (the
graph representation on the left).
b. If you compare substrate w/out enzyme, the hill becomes MUCH larger. The enzyme helps adequately
c. Because of the DESTABALIZATION EFFECTS- you have a smaller hill to climb in order to reach the
transition state (graph to the right).
d. At the transition state the ES complex quickly rearranges itself to form more stable complex, electrons are
transferred to create the ultimate product, so part of this electron rearrangement is stabilized by the enzyme
a.a. As this happens, we create the second dip/hill back down to the EP complex- the enzyme with the
product.
e. This graph represents how a enzyme assists in getting to through the transition state, and ultimately the final
product.
VII. How does destabilization of ES affect enzyme catalysis [S8]
a. Shows binding event
b. Substrate molecules are twisting, vibrating all over the place constantly. So that when they come into bind
they might be a little twisted in charges within the active site will bind to the substrate. This can sometimes
lead to vibrations between the molecules. These factors cause significant strain- an imperfect fit.
c. The enzyme can often help to change conformation when the substrate comes in.
d. All of the factors cause STRAIN & ultimately raises the energy within the first step of the reaction.
VIII. How does destabalization of ES affect enzyme catalysis [S9]
a. substrates, proteins, and the binding pocket are often surrounded by multiple sheets of water
b. upon binding, we must desolvate…which requires energy. This also helps to destabilize the process.
c. This process raises the energy of the ES complex, making it more reactive- the reaction rate will inc.
IX. How does destabilization of ES affect enzyme catalysis [S10]
a. Illustrates charge interactions between the substrate and the enzyme
b. Substrate binding to enzyme initially, is usually NOT favorable-because it was stripped of water & it now is
being repelled from the similar negative charges within the binding pocket
c. ALL THREE DESTABLIZATION FACTORS ARE VITALLY IMPORTANT IN THIS PROCESS
X. How tightly do transition-state analogs bind to the active site [S11]
a. The affinity of the enzyme for the t.s. can be 10x-20/ 10x-26. This is very tight binding
b. Its very unusual to see reactions with these kind of properties in a test tube
c. These transition states are chemically and physically stable
d. Can make derivatives of natural substrates ANALOGS, something that looks similar in structure but has
different substituents bound to it. Refers back to slide 10- with the negative & negative charges…exchange
one of the charged groups with an uncharged group to allow the bond to be significantly tighter. This process
is done very frequently with drug research. Scientists use the structure of the enzyme & what it naturally
interacts with and start developing the drug from there.
e. Take known natural substrate & place it in a columnadd substituents to the molecule to change one/two
aspectsmake thousands to test & see which ones binds with more affinity
f. Goal of t.s. analog= making something that has an even higher affinity in the transition state, so that the
enzyme prefers to bind to the analog, rather than the substrate.
g. You can inhibit enzymes in this fashion-using an analog. It will prohibit the enzyme from interacting with the
natural substrate.
FUNDAMENTALS 10:00-11:00
MONDAY, AUGUST 23rd, 2010
DR. LARRY DELUCAS
ENZYME CATALYTIC STRATEGIES
Scribe: BO BRADFORD
Proof: ROSS ISBELL
Page 3 of 5
XI. How tightly do transition-state analogs bind to the active site [S12]
a. Example of a conversion of a natural substrate into its analog.
b. Look similar, but have small changes: a charge, another carbon atom, different stereochemistry
c. Have higher binding affinity for natural transition state than the normal substrate
XII. How tightly do transition-state analogs bind to the active site [S13]
a. Another example
b. NOTICE CHANGES IN STRUCTURE & AFFINITY
XIII. How tightly do transition-state analogs bind to the active site [S14]
XIV.
Transition state analogs make our world better [S15]
a. Enzymes are often targets for drugs and other beneficial agents
b. Make ideal enzyme inhibitors
c. EXAMPLES:
i. ENALAPRIL- lowers blood pressure
ii. STATINS- lower cholesterol
iii. PROTEASE INHIBITORS- AIDS drugs, in this case they did have the structure, but still started with the
natural structure. The drug today, closely resembles the natural substrate
iv. TAMIFLU- viral neuraminidase inhibitor. (spoke about his “spin off company” developing drugs)
XV. Transition state analogs make our world better [S16]
a. Picture from book with different drugs
XVI.
Transition state analogs make our world better [S17]
a. Example of another analog
XVII.
Transition state analogs make our world better [S18]
a. Lipitor, a statin, treats people with high lipids/cholesterol
XVIII.
Transition state analogs make our world better [S19]
a. Usually these analogs are hydrophobic in character and water soluble
b. Typically, analogs have molecular weight that is less than 500
c. Can be manufactured as peptides, but if so- they are usually degraded readily within the stomach.
XIX.
Transition state analogs make our world better [S20]
a. No comments
XX. Transition state analogs make our world better [S21]
a. Doesn’t just apply to humans.
b. Can also use micro-organisms from plants or other organisms
c. They have different proteases or enzymes within them that are capable of being blocked.
d. If blocked- the lyse cycle is stopped. Allowing the opportunity to get rid of the bug
e. Some can be sprayable as insecticides; in human diseases, scientists can synthesize a transition state
analog for a protein to block the lyse cycle of a micro-organism- to block replication
XXI.
Transition state analogs make our world better [S22]
a. Example in the book of a transition state analog
XXII.
Tamiflu is a viral neuraminidase inhibitor [S23]
a. Drug differs significantly from Dr. Delucas’ model.
b. Both have aromatic rings, but his “spin off company” version doesn’t have a carbonyl group nor the ester
group.
Tamiflu is a viral neuramindase inhibitor [S24]
a. This molecule has two enzymes on the exterior: 1) Hemagluttinin 2) Neuraminidase
XXIII.
FUNDAMENTALS 10:00-11:00
Scribe: BO BRADFORD
MONDAY, AUGUST 23rd, 2010
Proof: ROSS ISBELL
DR. LARRY DELUCAS
ENZYME CATALYTIC STRATEGIES
Page 4 of 5
b. The head of the neuraminidase is a homotetramer. The active site of this enzyme contains 11 amino acids
that have never changed throughout all strains of the flu.
c. Drug blocks & interacts with the 11 a.a.
d. This conservation of these a.a. within the active site, allows scientists to target this area for drug research
easily.
e. Within active site the 11 a.a. never change-other amino acids within the protein mutate frequently
XXIV.
XXV.
XXVI.
XXVII.
XXVIII.
XXIX.
XXX.
XXXI.
XXXII.
XXXIII.
XXXIV.
XXXV.
Tamiflu active site domain picture [S25]
a. Magnified schematic diagram of the active site of a protein associated with the protein
b. Notice the conserved amino acids within the site
Homotetramer schematic diagram [26]
a. Very dynamic molecule that is always open and closing
b. Notice its’ globular shape, with the small purple active sites within each domain
How many other drug targets might there be? [27]
a. This information is significantly out of date, and not accurate with present day statistics
b. More than 3000 experimental drugs under study
c. These and and many future drugs will be studied using the transition-state analog techniques
How to read and write mechanisms [S28]
a. Lewis dot theory is used to read & write reaction mechanisms
b. Electronegativity allows us to determine where shared electrons will be residing during a specific period of
time: F > O > N > C > H. This is very important with reaction mechanisms & the formation of bonds
How to read and write mechanisms [S29]
a. A curved arrow represents the movement of an electron PAIR (from a filled orbital to an empty one)
b. A full arrowhead represents an electron PAIR
c. A half arrowhead represents a SINGLE electron
d. To interpret a Bond-breaking event, the arrow begins in the middle of the bond
How to read and write mechanisms [S30]
a. This example shows a bond being broken & two electrons going to substituent B
b. As a result the B takes on a negative charge due to its valence electron while the A becomes positively
charged
How to read and write mechanisms [S31]
a. This example shows an arrow starting at the sourc of the electrons and points to the atom where the new
bond will form
How to read and write mechanisms [S32]
a. This example shows a proton transfer due to a nucleophillic attack
b. A nucleophile has an extra pair/one electron that attacks another atom which ultimately shares the extra
electrons
How to read and write mechanisms [S33]
a. A proton transfer can change a nucleophile into an electrophile, and vice versa- at first the nucleophile
wanted to share electrons, but afterwards it needs to accept more
b. Thus, we should consider that the protonation of substrate active-site residues & how the pKa values can
change in the environment of the active site
c. Amino acids that have specific charges, have the capabilities to share electrons
d. HIS- has a pKa with a pH close to physiological pH-this means that a percentage of its hydrogen atom is
ALWAYS COMING OFF. This is why HIS is always involved with many chemical reactions
How to read and write mechanisms [S34]
a. Diagram of HIS in a chemical reaction
b. Hydrogen atom on HIS residue undergoes nucleophillic attack from B
c. As the nucleophile interacts with the hydrogen, the electrons end up getting transferred to the nitrogen
d. And the hydrogen is transferred to the oxygen atom making another bond
How to read and write mechanisms [S35]
a. Another diagram of a chemical reaction
b. Oxygen interacts with carbon, forcing the double bond on the carbon to force two electrons up to the oxygen
atom giving it an partial negative charge
What are the mechanisms of catalysis
a. Enzymes facilitate formation of the near-attack complex
b. At times the enzyme & substrate come together and join with another substituent. The enzyme facilitates this
process by positioning charged groups/sterically hindered groups in distinct locations.
FUNDAMENTALS 10:00-11:00
Scribe: BO BRADFORD
MONDAY, AUGUST 23rd, 2010
Proof: ROSS ISBELL
DR. LARRY DELUCAS
ENZYME CATALYTIC STRATEGIES
Page 5 of 5
c. When reactions occur, components are within van der waals distance from one another- VERY TIGHT FIT
d. This allows for a quick interaction, because everything is in such proximal location
e. Protein motion is essential to enzyme catalysis. Enzymes can change its conformation to allow proper
interaction with substrate.
f. General acid & base catalysis where electrons/ charges are transferred
g. Covalent interactions can also occur, as well as low barrier H-bonds
h. H-bonds with similar electronegative groups, the electrons between the two can be shared equally b/t themthus the barrier of the electron to leave one to go to the other is MUCH lower. This technique is used in the
TUNNELING OF ELECTRONS
i. Metal ions can also be used in catalysis
XXXVI. Enzymes facilitate formation of near-attack complexes [S37]
a. X-ray crystal structure studies can show us how closely related these interacting groups are to one another
b. Pre-organization selects substrate conformations in which the reacting atoms are in van der waals contact
and @ an angle resembling the bond to be formed within the transistion state.
c. Thomas Bruice termed these arrangements NEAR ATTACK CONFORMATIONS (NAC’S)
d. NAC’S= Precursors to reaction transition states
XXXVII. Enzymes facilitate formation of near-attack complexes [S38]
a. Without an enzyme, the probability of two reactive molecules coming together in a specific conformation is
INCREDIBLY SMALL.
b. Enzyme plays tremendous role in taking an improbable situation and converting it into a probable situation
within the active site.
XXXVIII. Enzymes facilitate formation of near-attack complexes [S39]
c. Diagram interpreting the energy barrier that must be overcome without an enzyme
d. The NAC positions the substrate in a specific place, making the creation of the transition state easy-the
energy barrier is much lower allowing for the reaction rate to be much higher.
XXXIX. The active site of liver alcohol dehydrogenase- a near attack complex [S40]
a. Picture from the book
b. An example that shows how the amino acids interact through hydrogen bonding or charges
XL. Proteins motions are essential to enzyme catalysis [S41]
a. Again- the movement of a protein is vitally important- enzymes depend on this
b. Movement allows the substrate to enter the active site of the enzyme as well as helping with the
rearrangement of the transition state to eventually allow the reaction to occur
c. The protein motion support catalysis in several ways
i. Assist substrate binding
ii. Bring catalytic groups into position
iii. Induce formation of NAC’S
iv. Assist in bond making/breaking
v. Facilitate conversion of substrateproduct
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