Mechanisms Flashcards
How do biological catalyts differ from ordinary chemical catalysts
higher reaction rates
milder reaction conditions
greater reaction specificity
capacity for regulation
Overview of a structure and function of a biological catalyst
most are proteins, although ribozymes exist
may require a co-factor ( such as inorganic ions, organic or metalloorganic molecules (coenzymes)
can be extremely specific for a substrate, and may even be stereospecific
Cofactors
Any factor required for enzyme activity or protein function.
Cofactor = inorganic ions
Coenzyme = vitamins, or a protein, can be further divided into cosubstrates and prosthetic groups
prosthetic group
a tightly or covalently associated cofactor
Apoenzyme
protien part of enzyme
Holoenzyme
complete catalytic entity, including both the apoenzyme and the non-protien constituents
Substrate specificity
active site is geometrically and electonically complemntary to the substrate
permissive enzymes and theri substrates
example = chemotrypsin, a protease that can clease hydrophobic peptides that he N-terminus, however it may also clease esters
How do enzymes work tho
provide an envrionment within whihc a given reaction is energetically more favourable
Reactive site: pocket called “active site”
- where substrate is bound
- form enzymes-substrate complex
- plays a role in catalytic process
What do enzymes affect about a given reaction
They affect the reaction rate, NOT the position of the equilibrium
Collision therory of reaction rates
1 The particles must collide in order to react
2 Not all collisions result in a reaction, and the reactants must collide in the correct orientation.
- The reactants must possess a minimum energy, the activation energy to initiate the chemical reaction
Energy diagrams
Only colliding particles that are properly orientated can deliver kinetic energy into potential energy as least as large as delta G double dagger to be able to climb over the “hill” and produce products
Distribution of energies for ideal gases
note: only a small fraction of molecules have sufficient energy to react (overcome delta G double dagger)
also, at higher temperatures, more molecules possess the kinetic energy to overcome delta G double dagger, and the reaction rate increases
Effect of temperature on reaction rate
at approximately RT, a 10C increase in temperature increases a typical reaction rate by about 2-3 times
Rate =K[A]^n
where, k is temperature depended: K is only a constant if T= constant
Which order of reaction are most enzymes
first order
What is the purpose of the arrhenius equation
relates temperature to reaction rate and activation energy,
higher K = faster rate
HIgher delta G double dagger the lower the rate, and the higher the temp the faster the rate
What is the effect does delta G have on rate
delta G has no effect on rate
What is reaction rate linked to
delta G double dagger
Note: enzymes cannot force an unfavorable reaction to go in the forward direction, ie// enzymes do not affect the position of equilibrium
What determines the position of equilibrium
delta G Prime at stp,
thus the equilibrium constant is related to delta G prime STP for the overall reaction and not to the activation energy barrier delta G double dagger
How do enzymes solve the problem of the fact that particles need to collide in order to react
particles must collide in order to react, thus enzymes need to increase the frequency of the collisions
increasing the temperature, and concentration is not realistic for biological systems, however you can increase the local concentration in the enzyme active site.
How do enzymes solve the problem of the fact that reactants must collide i the correct orientation
enzymes can help to orient the reactants properly in the active site
How do enzymes solve the problem of the reactants needing to posses a minimum energy to overcome delta G double dagger
enzymes need to increase the number of reactants with the energy to overcome the delta G double dagger
the could increase the kinetic energy of the reactants (increase in temperature), but this is not realistic for biological sytems.
They can however lower the energy barrier for delta G double dagger
Catalytic stratagies commnly used by enzymes
procimity and orientation effects
preferential binding of the transition state complex
- binding energy
- induced fit
acid/base catalysis
covalent catalysis
metal ion catalysis
electrostatic catalysis
Prosimity effects
intramolecular reactions greatly increase the rate
the Fischer hypothesis
the Fischer hypothesis = lock and key
can explain specificity but not the efficiency
also to be noted, super tight binding can make it harder to stabilize the transition state if the substrate is bound too tightly
What is the purpose of favourable reactions preferentially with the transition state
special interactions with the transition state that are not seen with the substrate help to lower the activation energy of the transition state
What would happen if the enzyme only bound tightly to the substrate and not preferentially to the transition state
you would see a lower rate because if the enzyme bound specifically the substrate the reaction would be unfavourable, and moreover, it would also require energy to unbind the substrate.
What does the oxyanion hole stabalize in a serine protease
sp3 tetrahedral transition state
Why is energy required to bind a given substrate
enegy is required to de-solvate a substrate
Interactions involved in the desolvation of a substrate
in aqueous solution, substrates are associated with water molecules, this is also known as a hydration shell
must give this up when the substrate is bound to the enzyme active site
weaak interactions between the solvent and substrate are replaced with weak interactions between the enzyme and the substrate
-> will contribute to binding energy and facilitate catalysis
Delta G^R binding energy
refers to the favourable interactions between E and S upon forming the ES complex = “Michaelis Complex, before the formation of the TS
contributes to specificity as well as catalysis
release fo energy upon formation of ES complex might be released as head ( a local increase in temperature or KE to help achieve TS) or might be used to fuel conformational change accompanying the induced fit
Delta G^T binding energy
refers to interactions between enzyme and TS to stabilize TS and thus reduce delta G double dagger
Which type of binding energy is “energy saved” due to favouable interactions between E ad TS.
T bind
Since energy to achieve TS in the uncatlyzed reaction was never expended, there is nothing to release
energy saved
Whihc type of binding energy is “energy released” upond binding of S to E, due to favourable interactions
R bind
S had to give up favourable solvent interactions before this
driver reaction
What are the assumptions of the Kochland induced fit hypothesis
interaction is optimum between the enzyme and the TS (not the substrate)
Upon substrate binding to enzyme, the enzyme changes conformation in order to optimize the position of their reacting groups in order to favour catalysis
Applications of our understanding of “preferential binding of TS complex”
allows understanding and design of inhibitors
-> transition state analysis (TSA) are more potent inhibitors than substrate analogs
allows understanding/ design of enzymes
-> catalytic antibodies
Antibodies
the antibody will bind to the antigen via the Fab variable region, tagging the antigen, which will either impeded the activity of the pathogen or provoke th eimmune system to destroy the pathogen
catalytic antibody
normally antibodies have no catalytic activity
-> they do however bind to substrate, if it is a TSA is could in theory catalyze a reaction
Thus, a catalytic antibody is an antibody that has catalytic activity since it is raised against a TSA
- > demonstrates the importance of this mechanism of enzyme catalysis (preferential binding of the TS)
- > could assist int he design of new enzymes
Types of acid base catalysis
General acid cat
Gernaral base cat
concerted general acid-base cat
General acid catalysis
process in which partial proton transfer from a bronsted acid (a species that can donate a proton) lowers the free energy of a reactions TS
General base catalysis
process in which reaction rate is increased by partial proton abstraction by a bronsted base (a proton acceptor)
Concerted general acid-base catalysis
process in whihc reaction is simultaneously catalyzed by both processes
What kind of nucelophile is water
A bad one lol
Are oxyanions good leaving groups
no, they are terrible
Covalent catalysis
involves rate acceleration thorugh transient formation of a catalyst-substrate covalent bond
may provide an alternate, lower energy, path towards the formation of products
gers decrease in entropy to favour enyme catalysis; help in proper alignment of substrate in active site
What is the benifit of covlalent catalysis for the reaction of acetoacetate to acetone through a schiff bace intermediate
multiple resonace structutres and the imine acts as an electron sink
What are the three stages of covalent catalysis
1 nucelophilic reaction between catalyst and substrate to form covalent bond
2 withdrawal of electrons from the reaction centre by the now electrophilic catalyst
3 elimination fo the catalyst, though essentially the reverse of stage 1
Good nucleophiles
hydroxyl group
sulfhydryl group
amino group
imidazole group
good electrophiles
protons
metal ions
carbonyl carbon atoms
cationic imine (ie schiff base)
Metal ion catalysis
metalloenzymes = contain tightly bound metal ions (usually transition metals)
Metal-sctivated enzymes = loosely bind metal ions from solution (usually alkali and alkaline earth metal ions)
How to metals participate in enzyme mediated reactions
by mediating redox reactions through reversisble changes in the metal ions oxidation state
by binding to substrates to help orient them properly for reaction
*By electrostatically stabilizing or shielding negative charges
Give an example of a metal electrostatically shielding negative charges in an enzyme mediated reaction
formation fo Mg2+ complex in hexokinase
partially shields the negative charges and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP
metal electrostatically shielding negative charges in an enzyme mediated reaction as compared to protons
better, metal ions can be present at high concentrations at neutral or basic pH
Metal ionss can have charges > +1
Metal ions promote nucleophilic catalysis via water ionization
a metal ion’s charge makes its bound water molecules more acidic than free water, and can therefore serve as a source of -OH ions even below neutral pHs
example = human carbonic anhydrases
How does human carbonic anhydrase solve the problem of using water as a nulceophile
complex water to a metal ion such that hydroxide is used as a nucleophile
metal = Zn 2+ is chelated to histidines (3x) at the active site
this in turn increases the acidity of water (@ pH of 7)
What is the role of His 64 in human carbonic anhydrase
abstracts a proton from the zinc-bound water molecule. It is too far away to do it directly, however, two water molecules provide the proton via a proton shuttle
acts as a general base
What are the three classes of serine proteases
cysteine proteases
aspartyl proteases
metaloproteases
all three of these solve the same problem, but in different ways
Serine Proteases
Digestive enzymes
- chemotrypsin
- trypsin, both produced in the pancreas and released in the intestine
elastase, helps with tissue elasticity
enzymes of blood clotting cascades (serine protease)
and other closely related enzymes = serine esterases = acetylcholinesterase which cleases esteras and amide bonds??
Challanges faceing the serine proteases (and all enzymes for that matter)
proximity and orientation issues
recognition of substrate/ reactants
catalysis ( increasing the rate of reaction)
product release (product blocks active site, need to release quickly)
Must be responsive tot he needs of the cell/ tissue/ organisms ie// regulation
Which terminus do the serine proteases cleave at
C terminus
Which substrate does chymotrypsin prefer
bulky, hydrophobic amino acids
phy, trp, tyr
R = ser. neutral
Which substrates does trypsin prefer
positively charges amino acids
arg, lys
R = Asp - , binds positive groups
Which substrates does elastase prefer
small neutrall amino acids
ala
2x gly replaced with val 226 and the 216 which makes a super short and hydrophobic pocket
How the the serine proteases solve the substrate specificity problem
the specificity pocket
assists in substrate recognition
also helps to address the issue of proximity and orientation
Which challanges do the serine proteases face in terms of mechanisms of reaction
water is a bad nu
there is an unstable oxyanion int
NHR is a bad leaving group
these all contribute to the reason why proteins don’t spontationously degrade in water
How do the serine proteases solve the mechanistic problem
Thy catalytic triad, active site ser is involved in catalysis and hydrolysis
this improves the nucleophilicity, stabilizes the transition state via an oxyanion hole and also improves the quality of the leaving group
the specificity pocket is separate from the catalytic triad and helps with specificity and orientation
Role of Asp 102 in the catalytic triad of serine protease
improves the nucleophilicity of His57 via electrostatic interactions
Role of His 57 in the catalytic triad of serine protease
improvement of nucerophilicity of Ser195, its own nucleophilicity is improved by electrostatic interactions by Asp197
General base catalysis
in the next phase it acts as a general acid to improve the leaving group quality of Ser 195, b/c now R’-NH2 is a better leaving group
What is the role of Ser 195 in the catalytic triad of serine protease
Acid catalysic of the cleavage of a peptide bond. Acid-base cat via His 57.
ser 195 acts as a nucleophile, his 57 improves its nucleophilicity
also plays a huge role because it covalently attaches the substrate to the enzyme. (intramolecular reactions are faster)
also, through sn2 type rxn drives the transition state into the oxyanion hole
What happens is one member of the catalytic triad is removed / mutated
huge drop in rate, just as bad as all three mutated, removed
Why is the general rate of reaction higher in the fully mutated catalytic triad as compared to the uncatalyzed reaction
oxyanion hole stabalizes the transition state, and water could still diffuse in to act as a nucleophile1
What is the pursose of a huge enzyme given that a 2o amino acid loop is just as catalytically effective as a serine protease
might be specific, but cannot be regulated to suit the needs of the cell// tissue // organism
Why are enzymes so big?
to allow for proper formation of a catalytic site, and for the induced fit
also yenno regulation and allosteric control
Ketosteroid Isomerase (KSI), and site directed mutagenisis of R-Group H-bonds
Ki
wild type > pond mutant > conservative oxyanion mutant
in an artificial hydrophobic environment showed that a mutation of one of the R-groups that stabilized the TS decreased the rate by a log factor of 5-6
DIPF
DIPF-enz kills enz = serine protease
this is an irreversible inhibitor with covalent modification
mechanism based inhibitor = suicide substrate
thus, this is a good detector of a serine protease
Acetylcholineesterases
nerve gases, insectacides venoms and poisons, could also be used to treat alzheimers disease
can be irreversible inhibitors, many used therapeutically are indeed irreversible inhibitors
Competitive inhibitor
diffuses into active site and can leave
Suicide inhibitor
binds and mechanistically kills the enzyme
Concepts behind enzyme desgin
in theory, anything that stabilizes the TS could catalize a reaction
antibodies bind tightly to their antigens (= forms extensive noncovalnent interactons)
so, if an antibody could be designed that would bind a TS, it should stabilize the TS and catalyze the reaction
however TS are too unstable, so we could use a TSA to see if it would bind
what kind of nucleophile is CH2OH
hard
what kind of nucleophil eis CH2SH
soft
names of cysteine proteases
papin
caspase-1
Hepatitis C virus peptidase 2
cathepsin K
What type of active site to cysteine proteases utalize
catalytic diade
Carboxypeptidases
Enzymes that hydrolyze peptide bonds at the C-terminus end of a protein or a peptide
includes serine, cysteine, and metallo-carboxypeptidases
Carboxypeptidase A
A for aromatic
prefer to cleave peptid ebonds of C term with side cahins that contain aromatic or branched hydrocarbon chains
Carboxypeptidase B
B for basic
prefer to cleave peptide bonds of C term with side chains that contain positively charged amino acids
ex lysine and arginine
Panceratic exopeptidases: CPA1 and CPA2
A carboxypeptidase A
critical for may processes in the body including digestion, post-translational modification of proteins, blood clotting and reproduction
Active site of carbozypepidase A
single polpeptide with active site zinc
Zn2+ coordinated with two His and one Glu
Conformational change of Carboxypepidase A (and other enzymes) is important for
Induced fit
Tyr 248 moves and swings when the substrate binds
closes active site cavity
completes conversion from water-filled region into a hydrophobic one
Induced fit
catalytic groups of the enzyme are brought into the correct orientation by the binding of substrate
Kinases and induced fit
hexokinase
Kinases catalyze the transfer of gamma-phosphoryl of ATP to R-OH
Problem= need to prevent transfer to water
would be devastating to catalyze simple hydrolysis of ATP instead of a phosphoryl transfer
problem solved with an induced fit
examples of Aspartic proteases
pepsin chymosin Cathepsins Renin beta secretase HIV-1 protease
Pepsin
digestive enzyme found in the stomach
broad specificity: will highest 20% of ingested amide bonds
cleaves preferentially on N terminus of hydrophobic preferably aromatic AA residues
How are we attempting to treat HIV
aspartyl protease
HIV protease structure
is a dimer, with a single active site formed by two identical symmetrically arranged subunits
active site aspartic acid residue
flaps close down after substrate bound
