BCHM-4310 Study Guide - Final Guide: Relative Permittivity, Intramolecular Reaction, Serine Protease
Enzymes
I. Catalytic Mechanisms
a. Metal Ion Catalysis
i. Metals used: Fe2+, Fe3+, Zn2+, Mg2+, Mn2+, Ca2+, Co2+
ii. Gel shift analysis of inosine substrate
1. Let enzyme bind to DNA and run on gel
2. Now add different metal to see how bands change
3. Without metal, enzyme is unstable and can’t bind to DNA
4. Metal causes binding and a slow moving band
iii. Electrophilic catalysis
1. Stabilizing or shielding negative charges developed during transition state
2. Metal ions help stabilize charge density
iv. Metal ions are sources of Nucleophilic OH- at neutral pH
1. Metal bound to water decreases pKa providing more hydroxyls to reaction
2. Example is carbonic anhydrase
3. Generates bicarbonate
4. Zn ion coordinated by 3H and fourth ligand provided by H2O
a. Water is deprotonated which will attack CO2
v. Mediation of oxidation-reduction reactions
b. Electrostatic Catalysis
i. Binding of substrate excludes water from active site
1. Local dielectric constant is same as organic solvent where electrostatic
interactions are much stronger
ii. Charge distribution about active site is arranged to stabilize transition states
iii. Charge distributions serve to guide polar substrates toward binding site so rate of
reaction is greater than their diffusion-controlled limits
c. Catalysis through Proximity and Orientation Effects
i. Studied by William P. Jencks
1.
2. Experiment with reactions of carboxylates with substituted phenyl esters
3. Synthesized a series of compounds and measured reaction rate
a. Changed from intermolecular to intramolecular reaction
b. Continued restriction of flexibility and keeping substrates close
increases rate
4. Effective molarity (EM or EC)
a. EM = k1(s-1)/k2(M-1s-1)
b. Effectively shows how much you need to increase concentration of
lone substrates to reach catalyzed rate
d. Transition State Binding
i. Energy of substrate binding
1. In order for enzyme to work well, it doesn’t have to bind very tightly
a. If ES is too stable, reaction won’t complete because activation
energy will still be high
1 M- 1
s- 1
220 s- 1
5.1 x 104 s- 1
2.3 x 106 s- 1
1.2 x 107 s- 1
220 M
5.1 x 104 M
2.3 x 106 M
1.2 x 107 M
Entropy
Relative rate
(krel)
Effective molarity
(EM)
find more resources at oneclass.com
find more resources at oneclass.com
b. Enzyme needs some affinity, but it should involve binding to
transition state
ii. Serine protease
1.
2. ES has substrate in trigonal conformation and transition is tetrahedral
3. Oxyanion hole becomes filled
iii. Transition-state (TS) analogs
1. Transition-state analogs are stable compounds whose structures resemble
unstable transition states, very short lived (around 10-30 seconds)
2. 2-phosphoglycolate, a TS analog for trios phosphate isomerase
a. TPI catalyzes rapid aldehyde-ketone interconversion
iv. Catalytic antibodies
1. These are also called abzymes
2. Antibody-catalyzed amide hydrolysis
a. TS analog antigen
b. Reaction catalyzed by catalytic antibodies
c. Antibodies have high affinity to TS analog and can act as enzymes
II. Enzyme Kinetics
a. Rapid Equilibrium Kinetics
i. Studied by Michaelis and Menten
1. Henri also made significant contributions
ii. Un-catalyzed reaction
1.
2. v=k[S]
iii. enzyme-catalyzed reaction
1.
2. V = ?[S] where ? is unknown k
iv. Rapid equilibrium allows you to look at first step in isolation from rest
1. Assume everything is always in equilibrium
2. Kd = [E][S]/[ES]
a. Kd is the dissociation constant
3. v = k2[ES]
4. mass law: [E]t = [E] + [ES]
v. rate calculation
1. v/[E]t = k2[ES]/([E] + [ES])
a. [ES] = ([S]/Kd)[E]
2. v/[E]t = (k2[S]/Kd)[E]/([E] + ([S]/Kd)[E])
a. cancel out [E]
3. v/[E]t = k2[S]/(Kd + [S])
4. v = k2[E]t[S]/(Kd + [S])
5. Michaelis-Menten equation: v = Vmax[S]/Km + [S]
a. Vmax = k2[E]t
b. Steady State Kinetics
i. Calculated by Briggs-Haldane
find more resources at oneclass.com
find more resources at oneclass.com
1. Substrate concentration is much higher than enzyme concentration
2. Substrate decreases and product increases
3. Free enzyme decreases and [ES] increases
4. Over long periods of time, [ES] will not change
a. Means that there is a constant turnover and that d[ES]/dt = 0
ii. Enzyme-catalyzed reaction
1. E + S <-k1-k-1-> ES ->k2-> E + P
iii. Steady state assumes [ES] remains the same
1. +d[ES]/dt = k1[E][S]
2. -d[ES]/dt = k-1[ES] + k2[ES]
3. +d[ES]/dt = -d[ES]/dt
4. [ES] = (k1[S]/k-1 + k2)[E]
iv. Rate Calculation
1. v/[E]total = k2[ES]/[E] + [ES]
2. Plug in [ES] in this equation
3. v/[E]total = k2[S]/(k-1 + k2)/k1 + [S]
4. V = k2[E]total[S]/(k-1 + k2)/k1 + [S]
5. Michaelis-Menten equation: V = Vmax[S]/Km + [S]
v. Rapid equilibrium vs. steady-state
1. The velocity equation derived from rapid equilibrium is a special form of
the steady-state equation
2. Km = (k-1 + k2)/k1 for steady-state
a. Km = Kd in rapid equilibrium
3. Assume k2<<k-1
a. Km = k-1/k1 = Kd
vi. Steady-State parameters:
1. Km
a. Constant derived from rate constants
b. Estimate of dissociation constant of E from S under rapid
equilibrium conditions
c. Equals the concentration of substrate needed for ½ maximum
velocity
2. kcat – measure of catalytic activity
a. number of substrate molecules converted to product per enzyme
molecule per unit of time, when E is saturated with S
b. values of kcat range from less than 1/sec to many million/sec
3. kcat/Km – catalytic efficiency
a. estimate of “how perfect” an enzyme is
b. apparent second-order rate constant that measures how enzyme
performs when S is very low
c. upper limit for kcat/Km is diffusion limit – the rate at which E and
S diffuse together
vii. Michaeilis-Menten equation
1. Each Vo vs. [S] point is from one kinetic run
2. Region A – vo = kcat[E]1[S]0
a. [S] >> Km
3. region B – vo = (kcat/Km)[E]1[S]1
a. [S] << Km
c. Linear Transformation
i. Linear plots of michaelis-menten equation
1. Lineweaver-Burk
2. Hanes-Woolf
a. Best because of smaller and more consistent error across plot
ii. Double-reciprocal Plot
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Dna: plasmid vectors, plasmids are small, circular dna molecules used as vectors for. Phosphodiesterase: endonucleases hydrolyze sites within a chain, ex. Isoleucine: synthesis, must be taken in through diet, synthesized in plants. Feedback inhibition: gene expression, substrate specificity of enzymes, commercial inhibitors, metabolic roles. Protein synthesis and turnover: source of oxidation in sk muscles, more energy efficient than glucose, cell signaling, activator for mtor protein synthesis pathway, glucose metabolism, facilitate glucose uptake by liver/sk muscles, physiological roles. Immune system: necessary for t lymphocyte growth, brain function, protein synthesis, neurotransmitter synthesis, and production of, muscle growth energy. Intramuscular signal transduction + nutrition -> anabolic effects of exercise: regulation of initiation of mrna translation -> regulation of protein synthesis - Leucine crosses bbb and metabolized into glutamate and glutamine, interfering with neuronal synapses and causing degenerative symptoms. Therapeutic treatments: dietary restriction of bcaas with supplementation on non branched.
