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Enzymology Exam| questions with Complete Solutions
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Enzymology Exam| questions with Complete Solutions
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Enzymology Exam| questions withComplete Solutions
Hammond's postulate - ✔Two states occurring consecutively after one another during
a reaction, having nearly the same energy content, will be similar in structure.
The TS will resemble the reactants, intermediates, or the products more depending on which
state is closer to it in energy.
Hammond's postulate ramifications - ✔If species are sequential on the rxn coordinate
and similar in energy, they are similar in structure.
Unstable intermediates on the rxn pathway are predicted to resemble the TS structure. The
structure of these can be obtained experimentally.
Changes in structure that stabilize or destabilize intermediates will do the same to TS's that lead
to them.
Applications of TS theory & Hammond - ✔- determining structure of TS can lead to design
of TS analogs with similar ground-state geometry to TS
- analogs can be used as enzyme inhibitors in drug therapy
- analogs can be used to determine mechanism of enzyme-mediates rxns
Analogs - ✔Structurally resembles transition state and stabilized due to bonds.
- unable to be acted upon since they have bonds that cannot be hydrolyzed
- good for irreversible inhibition
- higher activity seen with TS analogs than substrate analogs
EX. Chorismate mutase - ✔Chorismate - predominant in small organisms, used to
create amino acid residues.
,Generated various catalytic antibodies using different TS analogs as the antigen. Antibodies
will mimic the active site microenvironment to support the TS or substrate.
Steps in experiment:
1. Synthesize possible TS structure and substrate analogs
2. Animals are used to produce antibodies against each analog - each assigned a unique analog
3. Take antibodies and incubate with substrate in absence of enzyme
4. Document activity and product formation of each antibody
ABs were able to exhibit catalytic activity. Those resembling the TS closest had the most activity
and produced the most product.
Antibodies - ✔Hypervariable region - dictates the specificity or affinity for the molecule an
antibody was produced against. When raised against an analog, it will mimic the active site
of the enzyme the analog was for.
Antibodies raised against the most correct analog should show the highest catalytic activity, but
will not be as high as the enzyme since they are missing the catalytic residues.
Antibodies (cont'd) - ✔Are catalytically dead:
- have binding energy, desolvation effect, microenvironment, proximity effect, etc.
- have no catalytic residues
Testing analogs - ✔Add possible TS and substrate analogs to enzyme + substrate solution:
- will be able to determine inhibitory ability
- substrate will out-compete poor analogs
Mutating the enzyme:
,- will still have original active site and microenvironment
- but no diversity of active sites for testing, unlike with antibody production
Acid-base catalysis - ✔Occurs very slowly without an enzyme to stabilize transition state.
Concerns the donation or abstraction of protons from catalytic acids or bases.
Main amino acids that can act as acid-base catalysts:
- Asp, Glu, His, Lys, Cys
Acid-base rxns tend to be controlled by the pKa of an amino acid sidechain, which can be
altered through pH to alter catalysis.
pH-rate profiles can be used to distinguish between acid and base catalysis, lead to
identification of catalytic residues involved.
Altering pKas - ✔Use Glu or Asp:
- can lower pH in the microenvironment or alter pKa of specific amino acid residues
- create a repulsive situation that forces a residue to keep its proton and drive up the
pKa (usually when placed together)
Catalytic base - ✔Abstracts a proton during rxn. Active form is deprotonated form.
Increasing the pH will deprotonate more of the residue into the active form, increasing the
rate of rxn.
Catalytic acid - ✔Donates a proton during rxn. Active form is protonated form. Decreasing
the pH will protonate more of the residue into the active form, increasing the rate of rxn.
Electrostatic catalysis - ✔Charged transition state is neutralized by an oppositely-
charged group from the active site of the enzyme.
Main amino acids that act as electrostatic catalysts:
- Asp, Glu, His, Lys, Arg
, Electrostatic interactions in the active site (ion-pairs, salt bridges) can collectively attract
substrate into the active site pocket to stabilize the transition state to reduce the activation
energy.
Orotidine 5'-monophosphate decarboxylase - ✔Model enzyme:
- mediated by microenvironment
- positioning of CO2- group close to negatively charged Asp group so repulsion will aid in bond
breakage - ground state destabilization.
Phosphate is critical for proper positioning:
- binds to phosphate binding pocket through interaction with ordered H2O and
Arg * H2O provides stabilizing force with strong H-bond interactions
* Arg residue is conserved, uses positive charge to drive strong electrostatic interaction with P
- binding forces positioning of rest of substrate, forces CO2- close to charged Asp
Carbanion stabilization:
- may be through electrostatic interaction with a positive charged residue
- Lys may act as a catalytic acid to stabilize the negative C by shielding negative Asp charges or
donating a proton
Ground state destabilization - ✔Increase in energy, and decrease in stability, in going
from one state to another.
Sometimes occurs after enzyme-substrate binding.
Metal ion catalysis - ✔Specific type of electrostatic catalysis.
Uses positively charged metal ion to stabilize negative charges in the TS or intermediate to
increase the rate of catalysis.
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