PINAUD EXAM 2 Flashcards
basic characteristics of enzymes
increase rate by lowering AE
do not alter equilibrium constant
often require co-factors
usually proteins, sometimes RNA
highly specific to substrate and reaction
protease
catalyzes the hydrolysis of protein peptide bonds
thrombin
proteolytic enzyme in blood clotting
Cuts between Arg and Gly
trypsin
enzyme in the digestive system
cuts after Arg or Lys
holoenzyme
apoenzyme (inactive) + cofactor (coenzyme or metal)
∆G equation
∆G = ∆G° + RT ln [products]/[reactants]
Keq equation
kf/kr
3 ways to increase the rxn rate (k)
increase substrate conc.
increase T
decrease activation energy
ES complex characteristics
shape of active catalytic pocket is 3D (via steric hindrances of AA residues) and flexible, often nonpolar
induced fit: change conformation after binding
multiple weak interactions between E and S (H bonding, electrostatic, hydrophobic, VDWs)
transition state
short lived chemical state
highest peak of ∆G diagram
strong binding and flexibility of ES complex promotes formation of transition state
kinetic evidence for ES complex
rxn rate increases with increases substrate conc until a plateau (enzyme conc)
physical evidence for ES complex
x-ray crystallography
binding energy
some free energy released upon binding ES, helps form active site and lowers ∆G of transition state
enzymes speed up biochemical rxns by…
specific substrate recognition
multiple reactive steps at catalytic site
strong binding to transition state
efficient release of product
first order rxn
V = k[S], units s-1
second order rxn
V = k[S][B], units M-1 s-1
at low [S]…
Vo proportional to [S]
at high [S]…
Vo independent of [S]
Km
substrate concentration at 1/2(Vmax)
kcat
turnover rate (molecules/s), only works when Vmax has been reached
Michaelis-Menten equation
Vo = Vmax ([S]/[S] + Km)
what does Km say about the strength of ES complex?
low Km = stronger binding
high Km = weaker binding
enzyme efficiency measurement
kcat/Km
10^8 to 10^9 is catalytically perfect
lineweaver burke plot
reciprocal of Michaelis-Menten curve, linear
1/Vo = (Km/Vmax)(1/[S]) + 1/Vmax
types of reversible enzymatic inhibition
competitive, uncompetitive, noncompetitive
irreversible enzymatic inhibition
tight binding to enzyme
competitive inhibition
enzyme binds to S OR I
enzyme freed from I by increasing [S]
increases Km, Vmax unchanged
DHFR (dihydrofolate reductase)
needed for cell division
methotrexate (similar structure) = competitive inhibitor to DHFR, 1000x tighter binding, cancer drug
methotrexate
competitive inhibitor to DHFR (structurally similar)
1000x tighter binding than DHFR
cancer drug used to kill rapidly dividing cells
uncompetitive inhibition
I binds after S
ESI complex cannot make P
Vmax decreases and cannot be attained
Km decreases
High [S] does not overcome inhibition
noncompetitive inhibition
I and S bind at the same time
ESI cannot make P
Vmax decreases
high [S] does not overcome inhibition
4 types of irreversible inhibition
group specific modifying agent, affinity labels, suicide inhibitors, transition state analogs
group specific modifying agent
react with specific group at modifying site
affinity labels
inactivate enzyme by covalent modification
suicide inhibitors
chemical mechanism makes enzyme react covalently with inhibitor
transition state analog
similar to transition state structure, binds more strongly to E than S
proline racemase
enzyme that catalyzes isomerization of proline
pyrrole 2-carboxylic acid acts as transition state analog to planar proline ion (transition state)
penicillin
transition state analog and suicide inhibitor
inhibits glycopeptide transpeptidase (forms bacterial cell walls through peptide cross-linking)
statins
competitive inhibitors of HMG-CoA reductase (cholesterol synthesis)
similar structure to substrate inhibits cholesterol synthesis
reactive cleft
environment favoring S + E interaction
close proximity of substrate to active reading groups
optimized orientation of substrate for rxn
rxn protected from water / hydrolysis
aspirin
irreversible covalent inhibitor of prostaglandin H2 synthase (prostaglandin synthesis –> transmission of pain info, inflammation)
acetylation of serine residue in channel to reach active site - anti-inflammatory
induced fit
stabilizes various conformations for both E and S
optimized orientation of catalytic groups in enzyme
allows very tight binding to transition state (gives free energy to accelerate catalysis)
covalent catalysis
reactive groups of enzyme become covalently attached to substrate
covalent E-S bond highly reactive for next step
usually involves strong Nu-
acid-base catalysis
reactive groups of enzymes donate or accept a proton
involves acidic and basic AA residues
metal ion catalysis
loosely (Ca2+) or tightly (Zn2+) bound to enzyme
ionic interactions with substrate or enzyme groups
shield neg charges or stabilize charges
serine protease
uses Ser 195 as a highly reactive group for catalysis
transient covalent interaction
acetylation & deacetylation
oxyanion hole
area of active catalytic site that tightly binds tetrahedral transition intermediate, stabilizes O- charge
catalytic triad
making Ser 195 a Nu-
H-bound network between Asp 102, His 57, Ser 195
mutations within triad lead to dramatic decrease in catalytic efficiency
chymotrypsin
cuts after bulky hydrophobic AAs, Trp, Phe, Met
pocket is deep and hydrophobic
trypsin
cuts after long positive AAs Lys and Arg
Asp (-) at the bottom of pocket
elastase
cuts after AAs with small side chains, Ala and Ser
narrow pocket
cysteine protease
catalytic mech resembles Ser triad
Aspartyl protease
use Asp carboxylate group to activate H2O and attack peptide bonds
Metalloproteases
use metal ion to activate H2O
ex. carbonic anhydrase
proton shuttle
His 64 removes proton in carbonic anhydrase catalysis to achieve fast catalytic rates
“committed step”
first step that makes reaction irreversible, feedback regulation will target the product of the first committed step
homotropic effects
caused by substrate itself at catalytic sites
increase catalytic rates
heterotropic effects
caused by binding of non-substrate ligands
decrease catalytic rates
R + T states
relaxed and tense states of an enzyme, rapid switching
cooperative binding
binding of 1 substrate causes increased binding affinity for another
ATCase
allosteric enzyme involved in synthesis of pyrimidine nucleotides
end product for ATCase?
CTP, which causes negative feedback on ATCase
structure of ATCase
12 subunits: 6 catalytic and 6 regulatory
PALA
substrate analog, binding to ATCase causes large conformational changes
equilibrium toward R state
(basically a model to understand substrate binding and how it affects catalytic activity)
CTP
allosteric inhibitor of ATCase
binds to regulatory subunits in T state, stabilizes low catalytic efficiency, equilibrium toward T, decrease ATCase affinity for substrates
ATP (in ATCase)
allosteric effector of ATCase
competes with CTP on regulatory subunits
favors R state and pyrimidine synthesis
isozyme
multiple forms of an enzyme catalyzing the same reaction in different ways or in different tissues
long-term regulation (M and H isozymes of LDH based on aerobic conditions throughout lifespan in rats)
kinase and phosphatase
kinase > phosphorylates (transfers phosphate group from ATP to AA residue), signal amplification
phosphatase > dephosphorylates (hydrolysis of phosphate ester)
cascade amplification effects!
Protein Kinase A (PKA)
phosphorylates many proteins!
2 C 2 R subunits
activated by cAMP
PKA consensus sequence
Arg - Arg - small AA - Ser/Thr - large AA
PKA substrates
1) consensus seq / pseudosubstrate (inactive)
2) ATP (phosphorylation cascade)
PKA shabangle
cAMP binding > conf change > frees C subunits (pseudosubstrate) > activates PKA > binds ATP > phosphorylation cascade
amplification example
1 cAMP causes a lot of phosphorylation
adrenaline causes a lot of G-protein to bind
zymogens
proenzymes/inactive forms of enzymes
activated by proteolytic cleavage
ex. proteolysis of trypsin activates chymotrypsin
reduction in sugars
hemiacetal - open - OH - reducing
acetal - locked - OC - nonreducing
non-reducing sugar
sucrose
reducing sugars
lactose and maltose
lactose
galactose + glucose
maltose
glucose + glucose
glycogen
glucose polymer
a-1,4 linkages (favor packaging), some a-1,6 for branching
reduce osmotic pressure
maintain glucose inside cell
cellulose
glucose polymer
B-1,4 linkages (fiber like structure for strength)
humans can’t digest these linkages
no branching
glycoproteins
protein modified by carbohydrate at specific AA residues via glycosyltransferase
ER and golgi
glycoprotein linkages
N-glycosidic linkage with Asn in Asn-X-Ser/Thr with X=any but proline
O-glycosidic linkage with Ser or Thr
erythropoietin (EPO)
blood glycoprotein involved in stimulating RBC production (higher number RBC, limit degradation)
recombinant EPO
helpful in treating anemia but also in blood doping
can be detected in blood due to different glycosylation pattern
blood types
based on glycosylation patterns
glycosyltransferase enzymes expressed by body define type
mucins
highly glycosylated proteins, act as lubricants in secretions (saliva, mucus)
Ser and Thr o-linked glycosylations
localized in VNTR domain
O-antigens
common oligosaccharide foundation
A-type glycosyltransferase
adds N-acetylgalactosamine
B-type glycosyltransferase
adds galactose
fatty acids
14-24 hydrocarbon chain w carboxyl
saturation of fatty acids
saturated = higher MP, less fluid
unsaturated = lower MP, more fluid
classes of membrane lipids
phospholipids (glycerophospholipids, sphingolipids)
glycolipids (sphingolipids)
cholesterol
glycerophospholipid
glycerol connected to 2 FA chains and PO4 - alcohol
phosphosphingolipid
sphingosine connected to 1 FA and PO4-choline
glycosphingolipid
sphingosine connected to 1 FA and mono/oligosaccharide
cholesterol structure
4 rings, 1 hydroxyl, 1 alkyl chain
parallel to other lipids with OH interacting with heads
arrangement of fatty acid chains
micelle: single fatty acid chain
liposome: closed lipid bilayer (circle)
formation driven by hydrophobic int, close packing by VDWs
liposomes
can be used as nanocontainers and reactors
thermodox: targets cancer tissue, heat releases toxins
permeability of lipid bilayers
low permeability to ions and polar molecules (except water)
highly selectively permeability barriers to ions using protein pumps/channels, maintains ion conc gradient
membrane protein functions
transport across membranes
signaling/transfer info
maintain electric potential
lipid : protein ratio in membranes
varies from 1:4 to 4:1
3 major types of membrane anchoring
transmembrane (very strong association)
electrostatic interaction (weak)
lipid anchoring (strong)
alpha helices
7-span proteins
20 AA/helix
extensive H-bonding
formed of hydrophobic AA residues
bacteriorhodopsin
light driven proton pump used by halobacteria, alpha helix
hydropathic index
uses ∆G when transferred from hydrophobic to aqueous environment
each point measures the AA window average ∆G (slides 1)
∆G > 0 hydrophobic
∆G < 0 hydrophilic
only detects alpha helices not B sheets!
alpha helix criterion level of +84
beta sheets
common for pore forming proteins
channel protein
extensive H-bonding, barrel structure
alternates between hydrophobic and philic AA
external: hydrophobic, internal: hydrophilic
prostaglandin H2 synthase
peripheral protein that converts arachidonic acid into prostaglandin in ER membrane
a-helix hydrophilic backbone for partial attachment, hydrophobic channel for arachidonic acid to get to active site
FRAP (fluorescence recovery after photobleaching)
shows that membrane is dynamic
after bleach, rapid lateral diffusion in the plane of the membrane for both lipids and proteins
recovery time as a measurement of membrane fluidity
what affects the overall melting temp of membranes?
length of fatty acids and degree of unsaturation
saturation takes priority over length
Tm
melting T of membrane (solid to fluid)
how does cholesterol influence membrane fluidity?
increases fluidity in low temperatures by disrupting tight packing
decreases fluidity in high temperatures by interfering with kinetic fatty acids
asymmetry in membranes
outer and inner membranes differ in lipid composition and associated proteins
maintained because flip-flopping is rare and requires flippase
orientation of transmembrane proteins do not change over time
outer membrane rich in glycosylated proteins and lipids
uncharged membrane permeable molecules
chemical concentration gradient determines spontaneous movement across membrane
charged membrane permeable molecules
chemical concentration gradient AND electrical potential term determines movement across molecule
∆G trans
determines if the transport across a membrane is passive or active
membrane pumps
primary active transport
drive thermodynamic uphill reactions with ATP
ATP causes conformational changes for open and close
membrane carriers
secondary active transport
drive thermodynamic uphill reactions using chemical gradient of one molecule to drive transport of a second molecule against own gradient
no ATP
membrane channels
passive transport
selective pore
can be sensitive to membrane polarization
What is SERCA ATPase: Ca2+ pumps
rapidly removes excess Ca2+ after Ca2+ triggered muscle contraction
needs ATP
P-type ATPase
10 transmembrane a-helices and 2 domains (A, P, N)
SERCA pumps mechanism
2 Ca2+ / ATP
1) 2 Ca2+ bind transmembrane domain from cytoplasm
2) ATP binds N domain
3) P domain phosphorylated on Asp residue
4) CC by A domain –> Ca2+ release into SER lumen
5) phosphoaspartate residue hydrolyzed (dephosphorylation)
6) transmembrane domain conformation reset
ABC (ATP binding cassette) transporter pumps
membrane pumps: 1 transmembrane domain and 2 ABC domains
Exist in equilibrium between open and closed conformations
Substrate binding stabilizes weak closed state, enhances affinity for ATP binding
ATP binding induces strong interaction between ABC domains > CC > substrate release
Reset of ATP pump by hydrolysis
types of cotransport
antiporter: 2 solutes in opposite directions
symporter: 2 solutes in same direction
no ATP
lactose permease symport
lactose permease in E. Coli uses H+ gradient to drive entry of lactose against
H+ binding favors lactose binding > CC > lactose release > proton release in cell
fastest transporters
1000 folds faster than pumps or carriers, close to diffusion rates of ions
action potential
generated in neurons by membrane depolarization involving Na+ and K+ ion channels
patch clamping
study ion channels by isolating single channel with a pipette, record changes in current upon opening and closing
ion specificity in K channel
1) sequence specific peptidic backbone TVGYG provides polar interactions with K+ as replacement for H2O, TVGYG optimal for K+ only and tight binding
2) electrostatic repulsions between K+ ion in channel allows for rapid flow
structure of K channel
tetramer forming pore through lipid membranes
larger solvated entry into channel, then narrow channel constriction forces passing K+ ion to desolvate (loos H2O) (funnel-like)
voltage gating
channel will open given a specific change in membrane potential
ligand gating
channel will open upon binding of specific ligand
voltage gating in K+ channels
S4++ domain at the bottom of the channel is positively charged
close channel: S4++ domain in the down position
open channel: membrane depolarization causes S4++ to go upward due to electrostatic repulsion, channel opens
ball and chain model
small peptide fragment is attached by a flexible domain, in open state it occludes the pore and inactivates transport
very quick, time depends on chain length
acetylcholine receptors
responsible for electrochemical signal transduction at synapses
binding of acetylcholine triggers opening of non-selective channel, Na+ and K+ travel freely, membrane depolarization (-60 to -20)
gap junctions
large channels
cell to cell communication
long opening times
controlled by membrane potential and phosphorylation
action potential mechanism
1) neuron firing > acetylcholine release in synaptic cleft
2) acetylcholine binding to receptors open ligand-gated channels
3) entry of Na+ and exit of K+ ions
4) at -40 mV, Na+ channels (voltage gated) open, more Na+ enters, further depolarization
5) Na+ channels inactivate, K+ channels open
6) K+ efflux repolarizes membrane, K+ channels inactivate
7) Na/K pumps use ATP to return to resting potential
aquaporins
rapid H2O transport
6 a-helices form hydrophilic channel
B-Adrenergic Receptor (B-AR) signal transduction
1) Adrenaline binds β-AR, activating receptor
2. Heterotrimeric G-protein (G⍺sβƔ) binds receptor and exchanges GDP for GTP
3. G-protein (β + Ɣ) subunits disassociate and G⍺s subunit binds downstream
effector adenylyl cyclase (AMP 1: 1 B-AR to many G⍺s)
4. Adenylyl cyclase converts ATP to cAMP (AMP 2)
5. cAMP activates PKA triggering a phosphorylation cascade and activation of other effectors (AMP 3)
Reset of G protein
hydrolysis of GTP to GDP
reassociation of G-protein units
reset of B-AR receptor
dissociation of ligand (adrenaline), phosphorylation of C-terminal tail allows binding of B-arrestin, desensitizes the receptor
angiotensin II receptor signal transduction
1) G⍺q-GTP binds, activates PLC
2) PLC binds PIP2, cleaves into IP3 and DAG
3) IP3 releases Ca2+ from ER, DAG activates PKC after it receives Ca2+
insulin receptor signal transduction
1) insulin binding > cross-phosphorylation by tyrosine binding on an activation loop
2) IRS bind receptor, bind PIP2 lipids with a pleckstrin domain, IRS phosphorylated
3) PIP3 Kinase recruited, makes PIP2 > PIP3
4) PIP3 activates PDK1
5) PDK1 phosphorylates Akt > cascade
6) storage of glucose via glycogen synthesis
calmodulin
Ca2+ binding domains called EF hands, large CC after Ca2+ binding
EGF receptor signal transduction
EGF stimulates cell growth
1) EGF binding > dimerization of receptor > CC
2) tyrosine kinase activity for cross-phosphorylation
3) recruitment of Grb-2, then SOS
4) Ras activated by GTP binding
Ras binds and activates kinases for P cascade
Ca2+ as an intracellular messenger
stored in ER, low cytoplasmic Ca2+, binding of Ca2+ to calmodulin = CC and signal transduction
adapter proteins
specialized domains (SH2, pleckstrin domain, SH3), needed for precise localization of signal transduction
kinase examples
PKA, PKC, PIP3K, PKB
receptors themselves can have kinase activity: insulin and EGF
second messengers
provide delocalization of signals in cell, fast amplification effects
Ca2+, cAMP, IP3
negative feedback mechanisms in signal transduction pathways
clocked deactivation (GTPase activity in G-protein, Ras)
general deactivation by phosphatase
ligand release of receptor internalization
Cancer and signal transdutions
gene encoding important proteins for signal transduction mutated
Ras gene often mutated, trapped in active form with lost ability to hydrolyze GTP > uncontrolled cell growth
cholera
toxin alters G-protein activity
Gas constantly active, continuous PKA activation
opening of Cl- channels
excessive loss of H2O and Na+, diarrhea/dehydration/death
myosin structure
two heads (motors), one long shaft
P-loop ATPase cores, 2 light chains
myosin substrate
ATP-Mg2+
myosin V ATPase activity
ATP hydrolysis by nucleophilic attack of H2O (takes off 1 phosphate)
vanadium replaces phosphoryl group
dephosphorylated = swinging lever-arm motion, power stroke state
actin binding of myosin, L T Head specific!
S1 heads (L and T) bind actin filaments
CC change favors release of Pi
1) T empty (strong actin binding) while L binds ADP + Pi (weak)
2) T binds ATP (detached)
3) ATP does to L head as ADP + Pi
ATP attachment and detachment only from T head
two models for myosin V movement
hand over hand: like a little human
inchworm: scoochies
structure breakdown of muscle fiber
thin actin filaments and thick myosin > sarcomeres > myofibrils > muscle fiber
each myosin filament binds 2 actin filaments on either end so able to contract
muscle contraction mechanism
1) ATP binds to myosin head, myosin releases actin
2) ATP > ADP + Pi, myosin head cocks to high energy conformation
3) Pi released from myosin, CC > myosin power stroke
4) ADP released
regulation of actin binding
1) action potential causes Ca2+ release
2) Ca2+ binds to troponin on actin, displacing tropomyosin
3) myosin heads can latch to actin
