Exam 2 Flashcards
3 ways to increase rate of reaction
- Increase temperature
- Increase substrate concentration
- add catalyst
Active site of serine protease
His 57- Acid/base catalyst
Ser 195- Nuc
Asp 102- hydrogen bond to stabilize
Chymotrypsin unique in that
it acts under mild conditions
Digests broad range of substrates
Oxidoreductases
oxidation-reduction reactions
ex: Alcohol Dehydrogenase
GAP dehydrogenase
Transferases
Transfer functional Groups
Ex: alanine aminotransferase
Hexokinase
Hydrolases
Hydrolysis (cleavage by H2O)
Ex: Chymotrypsin
Lyases
Group elimination, double bond formed
ex: pyruvate decarboxylase
Aldolase
Isomerases
Isomerization Reactions
Ligases
Bond formation coupled with ATP hydrolysis
Multiple enzymes catalyzing the same reaction
isozymes
What provides required catalytic groups when enzyme can’t
Cofactor
Metal ions
Can exist in multiple oxidation states, type of cofactor
Coenzymes
may be vitamins
- Co-substrates: enter/exit active site like substrate
- Prosthetic groups: remains in active site between reactions
Acid-base catalysis example
tautomerization of ketone
Acid-base catalysts
CLAGHT
Cys, Lys, Asp, Glu, His, Try
Covalent catalysis
Covalent bond between catalyst and substrate during transition state formation
Ex: Decarboxylation of acetoactetate (Schiff base), makes acetone
2 part reaction, 2 energy barriers with intermediate
Metal catalysis example
acetaldehyde to ethanol
Scissile bond
bond cleaved by hydrolysis is positioned near Ser 195 when substrate binds to enzyme
Why enzymes so large if few residues require?
Must precisely align active site
Transition state in chymotrypsin
low barrier hydrogen bond, transition state stabilized (energy lowered).
Bond between Asp 102 and His 57 becomes shorter as reaction goes on
Catalysis is effected by active site microenvironment. Explain this in hexokinase.
Electrostatic catalysis
nonaqueous active site allows more powerful electrostatic interactions between enzyme and substrate than aqueous solution
How are chymotrypsin, trypsin, and elastase evolved?
Divergent, common ancestor
How has subtilisin evolved?
Convergent: unrelated proteins have similar characteristics
Chymotrypsin preferences
cleaves peptide bonds following large hydrophobic residues
Trypsin preference
Arginine/Lys - basic residues
Elastase
Cleaves peptide bonds following small hydrophobic residues (ex: alanine, glycine, valine)
Controlling activity: Proteolysis
Inactive chymotrypsin —trypsin–> pi-C —> delta C —> alpha C
Last 2 steps by chymotrypsinogen
More active confirmation
Controlling activity: inhibitors
resemble substrate chemically, partial catalysis, but don’t complete reaction
Suicide inhibitor
antithrombin- covalent binding, shut off reaction
The fact that the enzyme physically combines with the substrate is suggested by
hyperbolic rather than linear curve
unimolecular
first order, depends only on one substrate
k= s^-1
bimolecular
second order, depends on 2 reactants
k = M^-1 s^-1
What two things does velocity concentration depend on?
substrate concentration and Km
Km- what’s going on and what does it indicate
substrate concentration when velocity is half max, indicates dissociation of ES
Shows how efficiently enzyme selects substrate and converts it to product
How do you get a linear Lineweaver-Burk plot
[S]»_space;> [E]
ES at steady state
Catalytic efficency
kcat/km
high kcat to maximize
Ability to convert substrate to product
units= M^-1 s^-1
kcat/turnover number
units = s^-1
Rate constant when enzyme is saturated with substrate
number of catalytic cycles that each active site undergoes per unit time
Limits to catalytic power
- electronic rearrangements during TS
- frequency of productive enzyme collision with substrate, max is diffusion-control limit
- Enzyme reach perfection when rate is diffusion controlled
Multi-substrate reactions
Km= each substrate is different. To find it, reaction velocity measured at different concentrations of one substrate while the other substrate is present at saturating concentration
Vmax = maximum reaction velocity when both substrates are present at concentrations that saturate their binding sites on enzyme
Random order mechanism
doesn’t matter who binds first
Ordered mechanism
one must bind first
ping pong mechanism
one binds, product released, then the other can bind
ex: transketolase
Multistep reaction
Ex: transketolase
intermediate: the 2 carbon fragment removed from F6P remains on enzyme while waiting for substrate
nonhyperbolic reactions
ex: hemoglobin
Oligomeric protein- cooperative binding, multiple active sites
Use quadratic equation
Allosteric attraction
irreversible inhibitor
any reagent that covalently modifies an amino acid side chain in a protein
ex: thymidylate synthases
Suicide substrate
enter enzyme’s active site and begin to react, just as a normal substrate would, but then gets stuck in active site
ex: 5-florouracil
Competitive inhibitor
Reversible
Substance that directly competes with substrate for binding to enzyme’s active site
Increase Km
ex: succinate dehydrogenase is inhibited by malonate
___ occurs when product of reaction occupies the enzyme’s active site, thereby preventing the binding of additional substrate molecules
Product inhibition
Substrate analogues make good inhibitors, but ____ make even better inhibitors
transition state analogs
Is rate of product formation = ES disassociation?
No, ES complex could be going backwards (k-1)
noncompetitive inhibition
Km binds to site on enzyme other than active site
Km=0
Kcat/vmax are DECREASED
ex: metal ions
Mixed inhibition
apparent Km may increase or decrease
Vmax decreases
Uncompetitive inhibition
multi-substrate reactor. inhibitor binds are one substrate has bound.
Vmax/Km are lowered to same degree, parallel lines
Allosteric inhibition in glycolysis: phosphoenolpryruvate.
Why inhibit primary regulatory step in glycolysis.
To prevent unneeded energy expression.
If pyruvate accumulates, too much glycolysis, not full use of product.
Feedback inhibitor and Negative effector
How does phosphoenolpyruvate inhibit glycolysis?
Swaps Arg 162 (+ charge) with Glu 161 (- charge)
F6P (- charge) no longer attracted to a + charge, is repelled instead
ADP
positive effector
binds to same sight as phosphofructokinase
forces ARG 162 to remain where ti can stabilize F6P binding.
4 factors that influence enzymatic activity
- change in rate of enzyme’s synthesis
- change in subcellular location
- ionic “signal”- change in pH, release of calcium
- covalent modification
Most modern drugs are _____. Ideal drugs are
inhibitors
high affinity, high selectivity, non-toxic
As Km increases
affinity of enzyme for substrate decreases
During inhibition if vmax isn’t 0 (as in competitive), it
Decreases
prosthetic group
cofactors that never leave molecule
What is prosthetic group in hemoglobin?
heme- iron in hydrophobic pocket between E and F.
6 bonds on iron
4 with nitrogen in ring
1 with histidine residue in F helix
1 with oxygen
Why must heme be part of a protein to be a good oxygen carrier?
by itself, central atom Fe(II) can easily be oxidized to FE(III), which can’t bind O2.
Myoglobin and hemoglobin: structural similarities
heme group in hydrophobic pocket
His F8 on iron
His E7 H-bond to O2
HOWEVER: primary linear structure different
Invariant residues
essential for function. If you change them, alter molecule function (similar in myoglobin and alpha/beta subunits)
Myoglobin and Hb are homologous proteins meaning
common ancestor (oxygen binding proteins)
Conservatively substituted residues
position under less pressure to maintain particular amino acid, can be substituted by similar amino acid
Variable position
position that can accommodate variety of residues, none critical for structure/function
Deoxy
Large cavity T P. ring is bowed low O2 affinity Heme Fe has 5 ligands
Histidine residue on beta between proline and threonine residues on alpha
Oxy
small cavity R heme group planar high O2 affinity Heme Fe has 6 ligands
Histidine residue on beta between threonine residues on alpha
Bohr Effect
reduction of hemoglobin’s O2 binding affinity when pH DECREASES ( [H+] increases)
BPG
stabilizes deoxy confirmation of hemoglobin to unload oxygen. Without, hemoglobin would bind too tightly.
5- charges that interact with + charge on hemoglobin
only binds to central cavity in T state
Fetus: H21 —> Serine, so…
+ charge that interacts with BPG is gone
reduces fetal binding, helps transfer O2 from maternal circulation to fetus
Microfilament functions
support PM determines cell shape structural support cell movement tensile strength
Actin polymer
polymerized is F-actin
+ end grows faster
ATP is - end
G-actin is globular
Prevent actin growth
adding capping protein.
Microtubule functions
reinforce cytoskeleton
construct cilia/flagella
align and separate chromosomes, form spindle apparatus
Microtubules structure
Binds to GTP/GDP
1 nucleotide binding site/tubulin subunit
Alpha GTP binding site buried in interface while beta exposed, GTP hydrolyzed, but resulting GDP remains bound and can’t diffuse
Single strand microtubule called
protofilament
Positive end of microtubule called
beta (alpha is negative, often anchored)
Paciltaxel
binds to beta tubulin
blocks depolymerization, stabilizes
Colchicine
destabilizes protofilament, interferes with side/side interactions.
Binds at interface between alpha/beta dimer
shut off cell division
Intermediation filaments: Keratin
coil-coiled.
dimer of alpha helices.
1/4th residues hydrophobic and holds coil together
7 residue repeat units
Keratin tetramers are antiparallel staggering held together by
cystine residues
Intermediate filaments: Collagen
Triple helix
every 3rd is glycine –> secondary structures
30% proline/hydroxyproline
Gly-Pro-Hyp triplet (left- more stable)
3 polypeptides in right hand triplet, stabilized by H bonding.
Staggered parallel
Where is collagen assembled?
Collagen is modified/cross linked by…
Endoplasmic Reticulum
covalent modifications
Myosin
microfilaments
change triggered by hydrolysis of ATP bound to head
unidirectional
alpha helix lever
Kinesin
microtubules
moves toward + end
unidirectional
Why is kinesin, unlike myosin, processive or highly processive?
kinesin constantly holds microtubules, many cycles of ATP hydrolysis and kinesin advancement occur before the motor dissociates from its microtubule tract.
Myosin dissociates after 1 stroke.
