Exam 3 Flashcards
Catalysts of biochemical reactions
Enzymes
Enzymes are _________ of biochemical reactions.
catalysts
What is a catalyst’s function
Increases the rate or velocity of a chemical reaction without being changed following the reaction
Most biological catalysts are __________
Proteins
Only exception currently known for biological catalysts
certain types of RNA molecules that can function as enzymes (ribozymes)
Enzyme concentration in cells
Very Low
Molecules that modulate enzyme activity
Effectors
Number of known enzymes
Over 1 million
Different species of organisms produce different __________ ___________ of the same enzymes
structural variants
Effectors can be either …
Activators or Inhibitors
Each enzyme has a
Specificity for a very narrow range of chemically similar substrates
Substrates bind to the enzyme at a region of the enzyme known as the
Active Site
The active site occupies
less than 5% of the surface area of the enzyme
________________ in the active site determines the type of substrate molecule that can bind and react there
The arrangement type of amino acid R-groups
There are usually about __ such R-groups per active site
5
Non-covalently bound _______ or covalently bound ____________ are non-protein molecules associated with enzymes at or near the active site
Cofactors (ex. Sugars, Lipids, Nucleic Acids)
Prosthetic Groups
Function of cofactors or prosthetic groups
Help to determine substrate specificity along with the enzyme’s active site
Enzymes are named according to…
the type of reaction they catalyze
Enzyme name =
4 integer EC number and a name
EC
Enzyme Commission: a group of expert enzymologists from all over the world who come up with the rules of nomenclature
1st integer of the EC number
indicates to which of the 6 major enzyme classes and enzyme belongs
2nd integer of the EC number
indicates the type of bond acted on
3rd integer of the EC number
indicates a subclassification of the bond type or group transferred
4th integer of the EC number
serial number
Enzyme that catalyzes the phosphorylation of D-glucose
Hexokinase
Why do we have EC numbers?
So that when you publish about a specific enzyme, people can use the exact enzyme
Catalyzes oxidation-reduction reactions
Oxidoreductase
A Hydrogen or electron donor is one of the substrates
Group Transfer A–X + B–> A + X–B
Transferase
Hydrolytic Cleavage of C–C, C–N, C–O, and others
Hydrolase
Non-hydrolytic cleavage of C–C, C–N, C–O, and others
Lyase
Results in a double bond or the addition to a double bond
Molecular geometrical rearrangement
Transfer of groups within a molecule to yield isomeric forms
Isomerase
Ligation (joining) of 2 molecules with accompanying hydrolysis of a high-energy bond (Condensation coupled to ATP hydrolysis)
Ligase
Activation energy is
the energy required for a reaction to proceed
Energy needed to drive a reaction
Energy Barrier
Binding the enzyme to the substrate ______ the activation energy
Lowers
If a reaction requires less energy, it is __________ to happen
more likely
When the enzyme binds to its substrate
Transition state
What is transformed into the transition state?
The substrate
Less energy is required for the reaction to proceed when
the substrate is in the transition state
The rate of a chemical reaction depends on
how efficiently the reactants can reach a transition state
If it does it easily we will have a high rate enhancement
The transition state is an ____________ ___________ arrangement of atoms in which bonds are being formed or broken
Unstable energized
Electrons are shifting when bonds are being formed and broken
The transition state is NOT a
reaction intermediate
Rection intermediates are stable structures formed as a result of a reaction series, so when it forms it is a stable molecule
Reactants are normally in the _______ ________ and have ________ potential energy for reacting
Ground State
Little
(Energy must be added to allow a reaction to occur)
The energy required to reach the transition state from the ground state is called the
Energy of Activation, or Activation Barrier, or Energy Barrier, or Activation Energy
Enzymes _______ the activation energy required to reach the transition state
Lower
The reaction is more likely to occur, thus the reaction rate is _______ if you lower the energy
increased
Circumstantial evidence comes from the use of molecules known as
Transition State Analogs
Stable structures that resemble the postulated (what we think) transition state structure
Transition State Analogs
Transition state analogs are _____ inhibitor enzymes
Powerful
If an enzyme has an analog stuck in the active site, the enzyme is now permanently bound to the analog and inactive, which makes the analogs such
Powerful Inhibitors
If we find a molecule that’s an analog of our transition state and we add that analog to our substrate the enzyme is going to…
bind to that transition state analog
Structurally the enzyme believes the analog is substrate. So now nothing happens because the analog is stable (there’s nothing left to happen) so the enzyme does not work.
The enzyme doesn’t release the substrate until
the product is formed (NOT when the transition state hits)
How to test to see if you have the correct transition state?
Look for molecules that are stable and are similar to the proposed transition state
Enzymes bind to analog and are no longer able to bind which…
inhibits enzyme activity
Mechanisms by which enzymes increase reaction rates
Facilitation of proximity, Covalent catalyst, Acid-base catalysis, and Molecular Distortion/Strain
Also known as the Propinquity Effect
Facilitation of Proximity
Reaction rate between 2 molecules is enhanced when the enzyme removes them from dilute solution and holds them in close proximity to each other in the enzyme active site
Facilitation of Proximity
This raises the effective concetration of reactants
An enzyme can increase the reaction between two molecules when…
an enzyme binds to them and takes them out of solution and holds them together
(Facilitation of Proximity)
A means to artificially raise reactive concentration so the reaction is faster
Facilitation of Proximity
Amino acids in the active site with nucleophilic R-groups attack electrophilic parts of the substrate forming covalent bonds between the enzyme and substrate
Covalent Catalyst
Nucleophilic groups include
COO- NH2, Aromatic OH, Histidyl groups, R-OH, S-
This mechanism is particularly evident in transferases (enzymes that most often use this mechanism)
Covalent Catalyst
Transfer of a proton in the transition state
Acid-Base Catalysis
Rate enhancement is only about a factor of 100 in this mechanism
Acid-Base Catalysis
Clu, His, Asp, Lys, Tyr, and Cys act as acid catalysts when they are protinated during this mechanism
Acid-Base Catalysis
When unprotinated the same amino acids can act as base catalysts
This mechanism is dependent on pKa of the R-groups that are in the active site and on pH optimum of the enzyme
Acid-Base Catalysis
Strain is induced in the bond system of the reactants and the release of the strain as the transition state converts to products provides the rate enhancement
Molecular Distortion/Strain
When the substrate is removed and converts into products
When the enzyme binds to the active site, strain is placed on the reactant
Molecular Distortion/Strain
Substrate in highly energized state, stressed state, enzyme lets go and that releases the energy in the transition state which helps push the substrate into product in this mechanism
Molecular Distortion/Strain
Discipline that describes the properties and characteristics of enzymes in mathematical terms
Enzyme Kinetics
When an enzyme is first mixed with a large excess of substrate there is an initial periods during which the concentration of ES complex builds up. It then…
finds substrate molecules and binds to them:ES
The enzyme complexed with the substrate
ES
The initial period where ES builds up is called
The pre-steady state
Usually too short to be easily observed
Pre-steady state
The reaction quickly achieves a ____________ in which the concentration of ES and the concentration of any other intermediates remains approximately constant overtime
Steady-state
Concerned themselves with the steady-state rate, and analysis of this type (referred to as Steady-State Kinetics)
Michaelis and Menten
Steady-state rate and analysis of this type
Steady-state Kinetics
Every time E converts S to a product, the enzyme binds to a new
substrate
Pioneer of science for women
Maude Menten
Enzyme + Substrate –>
ES–> E+P
E + S–>
ES k1
ES–>
E + S k1
Rate constant
k
The catalytic rate
Product formation rate = k2
Measures the number of S molecules turned over per enzyme molecules per second; number of substrates that get used for each enzyme in given time
k2
(Michaelis-Menton Equation) Vo=
Vmax[S] / Km + [S]
Every enzyme in the reaction is bound to a substrate
Enzyme saturation
If just on enzyme wasn’t bound to a substrate you would not achieve maximum product
Combined rate constant (Michaelis-Menten Constant)
Km
Total enzyme in reaction
Et
Maximum Velocity
Vmax
Initial velocity
Vo
k2[ES] =
Vo
Velocity increases with
increased substrate
A direct measure of _______ _________ can be judged by k2/Km
Reaction Efficiency (Enzyme Efficiency)
The theoretical maximum efficiency would fall between 10^8-10^9 s/mol/L assuming
every possible collision between E and S gives ES
Can also be directly assessed by k2/Km
Substrate specificity
Higher value for k2/Km if
enzyme is in solution with substrate it likes to bind with
Lower value for k2/Km if
Enzyme is in solution with substrate it doesn’t prefer to bind with
Almost a zero value for k2/Km if
enzyme can’t bind with substrate
Michaelis-menten Equation
Vo=Vmax[S]/Km + [S]
When Vo is 1/2 Vmax
Vmax/2 = Vmax [S]/ Km +[S] Km= [S]
A direct measure of _________ _________ can be judged by k2/Km
Reaction efficiency or enzyme efficiency
Theoretical maximum efficiency would fall between
10^8-10^9 assuming every possible collision between enzyme and substrate gives ES
Can also be directly assessed by k2/Km
Substrate specificity
Higher value for k2/Km if enzyme is in solution with…
Substrate it likes to bind with
It would be almost zero if it can’t bind with the substrate
Allows for a more accurate determination of Vmax using reciprocals and does not effect Km accuracy
Double reciprocal plots
Most popular double reciprocal plot
Lineweaver-Burk Plot
Reciprocal transformation of Michaelis-Menten equation
Double reciprocal plots
1/[S] on x-axis and 1/Vo on y-axis
Double reciprocal plots
Slope = Km/Vmax Y-int = 1/Vmax X-int = 1/Km
Reactions that usually involve the transfer of one atom or a group from one substrate to the other substrate
Bi substrate reactions
2 common mechanisms for bi substrate reactions
Ternary complex formation (random and ordered binding)
Ping-pong (aka double displacement)
Random or ordered binding mechanism
Ternary complex formation
Enzyme catalyzed before substrate comes in. Enzyme can only bind to S2 in its modified form. When enzyme binds to S2 it modifies it back to original enzyme.
Ping pong mechanism
Bi substrate reactions can be analyzed using
Steady state kinetics
Bi substrate graph with three separate starting points
Ternary complex formation
Bi substrate graph with parallel lines
Ping pong
