exam 1 Flashcards
(252 cards)
1
Q
water
A
life’s solvent
bent geometry
large electronegative difference
highly polar bc of large dipole moment
2
Q
hydrogen bonds
A
formed between water molecules to produce highly ordered and open structure
weak interaction
3
Q
surface tension
A
measure of internal cohesion
force needed to increase surface area
high surface tension in water
4
Q
coulomb’s law
A
quantifies interaction of ions in solution
5
Q
dielectric constant
A
measure of solvent’s ability to keep charges apart
6
Q
water as a solvent
A
ionization solvation (hydration) all interactions interferes with coulombic forces between ions high D (charges kept apart)
7
Q
hydrophobic effect
A
nonpolar molecules do not dissolve in water (hydrophobic, amphiphilic, or amphipathic)
ex. separation of oil and water
8
Q
water molecules in hydrophobic effect
A
water molecules more ordered around nonpolar molecule than elsewhere in solution
energetically favorable bc hydrophobic association releases water and increases entropy
9
Q
hydrophobic effect on proteins
A
protein folding–entails transition from disordered mixture of unfolded molecules to a comparatively uniform solution of folded protein molecules
10
Q
weak interaction
A
readily reversible, noncovalent interactions are essential biochemical properties (electrostatic interactions, van der waals)
allow transient, dynamic interactions and permit energy and info to move about cell
greatly affected by presence of water
11
Q
van der waals
A
stability in numbers over large surface of molecules
energy most favorable at van der waals contact distance
energy rises rapidly owing to electron-electron repulsion as the atoms move closer together than this distance
12
Q
is a higher or lower D value a better nonpolar solvent
A
lower
13
Q
amphiphilic and amphiphatic
A
same thing
molecule that contains both hydrophobic and hydrophilic groups
14
Q
how do insects walk on water
A
surface tension??
15
Q
why do soaps work against oily dirt
A
oily dirt surrounded by hydrophilic side of water molecules bc oil doesn’t dissolve in water
oily dirt washed away with water???
16
Q
would a globular protein fold correctly in a solvent of low dielectric constant
A
???
17
Q
how are all weak interactions fundamentally electrostatic interactions
A
???
18
Q
why are weak chemical bonds relevant in biology? and how is it advantageous to the function of some biomolecules
A
needed for DNA/RNA/protein synthesis
easily break hydrogen bonds of DNA and RNA in transcription and translation
19
Q
order of bond strength
A
???
20
Q
bronsted acid
A
h+ donor
21
Q
bronsted base
A
h+ acceptor
22
Q
acid base reactions
A
concentration of water is essentially unchanged
kw=ion product constant of water
ion concentrations are reciprocally related
23
Q
the strength of an acid is determined by what
A
dissociation constant (Ka)
24
Q
Ka
A
dissociation constant
larger Ka=stronger acid
25
strong acids
transfer all protons to h2o
| complete ionization
26
weak acids
little ionization
27
henderson-hasselbalch equation
pH= pKa + log([A-]/[HA])
28
buffers
mitigate pH change
| consist of acid base conjugate pairs
29
pK
midpoint
| pH at which acid and conjugae base are in equal amounts
30
buffering in blood
citric acid cycle produces CO2 as metabolite
| carbonic acid readily dissociates into conjugate acid base pair and acts as blood buffer
31
polyprotic acids
multiple pKs
one for each ionization step
steep equivalence points are pH jumps
32
is a solution with [H+]=10^-4 acidic or basic
acidic
33
how many pK values will be present in a titration of H3PO4
4
34
amino acid stereochemistry
chiral alpha carbon
| only L-amino acids found in proteins
35
what make D amino acids
racemases
36
amino acid properties
```
varied in:
polarity
acidity/basicity
aromaticity
bulk
conformational flexibility
ability to cross link
ability to hydrogen bond
chemical reactivity
```
37
amino acid use outside of proteins
metabolites
| energy sources
38
glycine
simplest AA
only non chiral AA
often in protein sequence for turns in a structure
39
hydrophobic AA
```
alanine
valine
leucine
isoleucine
methionine
proline
phenylalanine
tryptophan
```
40
alanine
utilized in glucogenesis
41
valine
leucine
isoleucine
branched aliphatic chains
42
methionine
provides CH3 to acceptor molecules in one-carbon metabolism
DNA transcription/translation begins with ATG (code for methionine)
important for production of red and white blood cells and platelets
43
proline
imino acid
closed chain
similar to glycine--found in turns of proteins
derivative= hydroxyproline (in connective tissue-makes stronger)
44
phenylalanine
alanine with benzene ring attached
necessary for synthesis of catecholamines (dopamine, epinephrine)
lack of Phe catabolism associated with phenylketonuria (PKU) and mental retardation
45
alanine
isoleucine
phenylalanine
branched hydrophobic side chains
| lack of polar surface area in side chain
46
tryptophan
used as fluorophore
precursor for seretonin--need trp to cross blood-brain barrier
in turkey and milk
47
psychedelic drugs
illegal hallucinagens of the tryptamine family
| mimic and interfere with neurotransmitters
48
uncharged polar amino acids
```
serine
threonine
asparagine
glutamine
tyrosine
cysteine
```
49
serine
alanine with OH
| activation site in enzymes regulated by phosphorylation, acetylation
50
threonine
similar to serine
also a target of phosphorylation in enzymes
acts as regulation site
51
asparagine
| glutamine
derivatives of aspartate and glutamate
| detox of ammonia, transport to live and kidney for urea synthesis
52
serine
| glutamine
increased polar surface area due to O and NH2 groups on the side chains
53
tyrosine
pka=10.46
precursor to dopamine and epinephrine (adrenaline)
derivative tyramine in cheese=pick me up
sometimes used a fluorophore
54
cysteine
pka=8.37
| forms disulfide bonds which are important for protein structure, connective tissues, hair
55
cystine
break disulfide bonds, shape, reoxidize
56
positively charged AA
lysine
arginine
histidine
57
lysine
pka=10.54
| connective tissue biosynthesis
58
arginine
pka=12.48
immediate precursor to urea
hypoargininemia associated with mental retardation
59
histidine
pka-6.04
precursor to histamine (signal molecule that tiggers allergic reactions)
anti-histamine used to block
60
negatively charged AA
aspartate
| glutamate
61
aspartate
| glutamate
pka=3.9
pka=4.07
neurotransmitter
62
aspartate
| lysine
charged side chains and polarity
63
essential vs nonessential AA
essential: obtain through diet
nonessential: synthesized in body
64
alpha-carboxylic acids
pka=2.2-3.5
65
alpha-amino groups
pka=9.4
66
pI
isoelectric point=1/2(pK1 + pK2)
| pH of net neutral charge
67
acidic AA
pI (net neutral charge) is between 2 lower pK's
68
basic AA
pI (net neutral charge) is between 2 higher pK's
69
formation of a polypeptide
carboxylic end attaches to amino end (amide bond)
water is released (condensation reaction)
dipeptide bond formed
70
tripeptide
3 bonds
71
oligopeptide
several peptide bonds
72
poly
more than 40 bonds
73
average AA residue
110 Da
74
polypeptide molecular mass
ranges from 40-3700 kDa
75
characteristics of a peptide bond
bond is not charged (side chains can be charged depending on pH tho)
bc they're uncharged peptide bonds can form tightly packed globular structures in proteins (not inhibited by charge repulsion)
76
chemistry of peptide bond
40% double bond character
shorter bond (increased rigidity)
trans position
flexible and fold
77
backbone of peptide chain
linked sequence of rigid, planar peptide groups
freedom of rotation on either side of peptide bond at pure single bonds
allows many different protein folds
78
phi and psi
measures of rigidity in the bond
as you look down bond undergoing rotation, phi and psi angles increase as 4th atom rotates clockwise relative to the first
79
why is 180 planar arrangement most stable angle in a polypeptide
steric interference at increased angle between adjacent residues
interaction between molecular orbitals
(side chain interference)
80
phi angle
angle between nitrogen of amino group and alpha carbon
81
psi angle
angle between alpha carbon and carbon of carboxylic group
82
are phi or psi angles more constrained
phi angles
83
factors that limit rotation
peptide bond planarity
side chain bulk
RESONANCE
84
residue
each amino acid in an oligopeptide
85
nomenclature of peptide bonds
start at N-terminus and end at C-terminus
| take off "ine" ending of each AA and add "yl" except for the last AA in sequence
86
what limits cis transformation in peptides
steric interference
87
what is the least constrained angle degree
180
88
laws of thermodynamics
1. energy can neither be created or destroyed
2. the universe tends toward maximum disorder
3. the entropy of a system approaches a constant value as the temperature approaches absolute zero
89
thermodynamics
describes energy flow
90
life and reproduction require
work
91
work requires
energy
| ex: synthesis of biomolecules, maintenance of concentration of gradients, movement of muscles
92
dynamic steady state
what we must maintain
| energy must be extracted, stored, and channeled
93
much of cellular biochemical apparatus devoted to
acquisition and utilization of energy
94
organisms
```
open system (exchange matter and energy with surroundings)
take up energy in the form of chemical fuels and sunlight (oxidize chemical fuels to extract their energy or absorb energy from the sun)
```
95
heat and work of a system
heat is absorbed BY the system FROM the surrounding and work is done ON the system BY the surroundings
96
change in energy equation
U=energy
| deltaU=q+w (heat + work)
97
cells
great transducters of energy
potential energy of chemical bonds in the fuel is converted to kinetic energy
convert chemical, electromagnetic, mechanical, and osmotic energy with great efficiency into heat and motion
98
cellular process of energy
potential energy goes into system in form of chemical fuels or sunlight
energy transductions accomplish work
comes out in the form of cellular work, heat, metabolites, or info-rich macromolecules
99
cellular work
chemical synthesis, mechanical work, osmotic and electrical gradients, genetic info transfer
100
metabolites
increased energy
| simpler than chemical fuels (CO2, H2O, NH3, HPO4 2-)
101
info-rich macromolecules
decreased entropy
simple compounds polymerize to form these
DNA, proteins
102
enthalpy
H
biological systems under constant pressure
change in enthalpy between initial and final states of process is heat generated or absorbed
exothermic: neg. change in enthalpy (heat is generated)
endothermic: pos. change in enthalpy (heat absorbed)
103
spontaneous process
characterized by conversion of order to chaos
104
entropy
S
disorder
+ deltaS= increased randomness
organisms are entropy poor and demand energy to create order
105
gibbs free energy
G
defined by enthalpy (H), entropy (S), and absolute temperature (T) in kelvin
deltaG=deltaH-TdeltaS
spontaneous: deltaG is neg. and energy is released
106
neg. deltaH and pos. deltaS
spontaneous at all temperatures
107
neg. deltaH and neg. deltaS
spontaneous only at low temperatures
108
pos. deltaH and pos. deltaS
spontaneous only at high temperatures
109
pos. deltaH and neg. deltaS
not spontaneous at any temperature
110
equilibrium constant
Keq
indicated tendency for reaction to go to completion
Keq=[C]^c[D]^d/[A]^a[B]^b
large Keq means reactants almost completely converted to products
111
how does deltaG related to standard free-energy change
deltaG=deltaG°+RTlnKeq
at equilibrium deltaG=0
expresses driving force of reaction
112
central dogma of molecular biology
DNA --> RNA --> protein
| transcription then translation
113
levels of protein structure
```
structure leads to function
primary
secondary
tertiary
quarternary
```
114
primary
unfolded amino acid sequence (denatured)
115
how long are most proteins
50-300 AA long
116
secondary
helix (antiparallel or parallel)
117
tertiary
one complete protein chain
118
quarternary
4 separate chains of hemoglobin assembled into oligomeric protein
119
alpha helix
```
ONLY helix with allowed phi psi angles AND H bond pattern (backbone)
right handed
3.6 residues per turn
5.4 angstrom pitch
rise=1.5 angstrom
tightly packed core
```
120
AA and the alpha helix
branched chain=steric clash (Val, Thr, and Ile)
AA's containing side chain H bond donors disrupt (Ser, Asp, and Asn)
Proline=alpha helix breaker
Gly found in turns
121
helices notation
n sub m
n=residues/turn
m=# of atoms in ring enclosed by by H bond
122
beta sheets
antiparallel (strands run in opposite directions)--hairpin
| parallel (strands run in the same direction)--crossover
123
left handed beta helix
tandem repeated sequence
124
fibrous proteins
secondary structure dominant
keratin and collagen are common fibrous proteins
2 stranded coiled-coil superhelix
125
keratin
```
extended coiled-coil structure
hair, horns, nails, feathers, claws
chemically unreactive
macrofibrils
coiled coil forms bc of 7 residue pseudorepeat in primary structure
```
126
superfamily "coiled-coil proteins"
myosin
| topomyosin
127
macrofibril
tiny part of hair cell
128
collagen
most abundant mammalian protein
Gly at every 3rd residue
Pro and hydroxylated Pro also common in sequence
OH added after protein synthesis by enzyme prolyl hydroxylase
OH groups confer stability
scurvy=lack of collagen
129
helix of collagen
GPP sequence directs formation of triple helix structure
helix crowded so Gly necessary (H bonds to main chain carboxyl)
well packed, rigid, strong
steric repulsion of pyrrolidine rings of P residues stabilize
130
globular proteins tertiary structure
3D fold of polypeptide chain
secondary structure spatial arrangement of AA residues far apart in sequence held together by forces including disulfide bonds
131
globular proteins
enzymes
transport and receptor proteins (perform chemistry in cell)
more intricate 3D structure
132
tertiary structure domains
structurally independent units of single polypeptide chain (sometimes separate function)
133
tertiary structure motif
common grouping of secondary structural elements
134
scientist that discovered alpha helix secondary structure of proteins
linus pauling
135
interior of globular proteins
hydrophilic residues
136
exterior of globular proteins
hydrophobic residues
137
protein folding directed by
AA interactions
138
how to proteins fold
spontaneously into native conformations under physiological conditions
folding is not random--directed by AA
structure stabilized by disulfide bonds
dialyze out denaturing agents
139
beta-mercaptoethanol
reduces disulfide bonds
| as disulfide bonds are reduced, beta-mercaptoethanol is oxidized and forms dimers
140
assisted disulfide bond formation
proper folding can be assisted by enzymes or other proteins
141
disulfide bonds
hold unstable state
| post-translationally modified proteins--proinsulin-->insulin
142
levinthal's paradox
discrepancy between actual time and calculated time it takes a protein to fold
143
answer to levinthal's paradox
cumulative selection
| tendency to retain partly correct intermediates
144
cumulative selection
complex proteins cannot switch from completely unfolded to completely folded
partly correct folds or intermediate conformations are stabilized (preserved) and the protein only has to seek out the best fold for portions not stabilized
145
what effects protein folding
secondary structure
| hydrophobic effect
146
secondary structure on protein folding
```
restricted conformations (helix and sheets)
level of specificity
steric constraints (compact polymers)
short range forces (H bonds, ion pairs, van der Waals)
```
147
hydrophobic effect
mutual exclusion of water
| residues predominate in protein interior
148
hierarchical organization
polypeptide chains form locally compact structures that associate with similar adjacent structures to form larger compact structures
149
folding funnel model of protein folding
folding funnel depicts thermodynamics of protein folding
depressions on the sides of funnel represent semistable intermediates that may facilitate or hinder formation of the native structure (depending on depth)
150
random conformational search model of protein folding
random conformational search=flat surface with narrow hole
| NOT CORRECT
151
classic folding model of protein folding
classic folding landscape=random search until "canyon" found that leads to native search
faster than random, but not fast enough
NOT CORRECT
152
landscape theory of protein folding
folding funnel=energy landscape
conformational adjustments to reduce free energy and entropy until native state reached
no local minima or transient energy barriers
153
molecular chaperones
heat shock proteins
| bind to hydrophobic surfaces (and release) and facilitate folding
154
obstacles to binding
solvent exposure
| aggregation with other molecules or itself
155
natively unfolded proteins
intrinsically unstructured proteins (IUPs)
bind to other molecule then form stable structure
more common in eukaryotes
molecular versatility important in signaling and regulation
156
amino acids not prevalent in natively unfolded proteins
V, L, I, M, W, Y (bulky)
157
amino acids prevalent in natively unfolded proteins
Q, S, P, E, K, G, A (polar)
158
protein misfolding diseases
bovine spongiform encephalopathy
alzheimer
parkinson
huntington
159
protein dynamics
atomic fluctuations: vibrations of individual bonds
collective motions: covalently-linked atoms move as units
triggered conformational changes
*fluctuations necessary for reactivity*
160
lack of structure provides:
more freedom to conform to and interact with a variety of proteins
161
assay for protein verification
assay for enzyme lactate dehydrogenase based on fact that product of reaction NADH can be detected spectrophotometrically
162
fractionation techniques
```
solubility
ionic charge
polarity
molecular size
binding specificity
```
163
clones
collection of identical organisms derived from single ancestor
164
recombinant DNA
insert DNA for your protein into an autonomously replicating DNA molecule (cloning vector)
165
host organism
what replicates DNA
166
cloning vector
circular, auto-replicating DNA "plasmid"
| cells become loaded with your protein
167
plasmids
extrachromosomal genetic element found in a variety of bacteria
has replication origin
168
transformation
laboratory technique that transfers plasmids to host bacterial cell (cells treated to be temporarily permeable to small DNA molecules
169
stringent vs. relaxed control in cloning
stringent tied to host replication
| relaxed can have much higher number of copies
170
restriction endonucleases
enzymes that allow recombinant DNA techniques
recognize and cut at palindromic sequences in DNA
twofold rotational symmetry
produces series of defined fragments which can be separated by gel electrophoresis
171
protein over-expression
vector inserted into host organism
cells of organism are grown
contain large amounts of protein
harvest cells by slow centrifugation to get wet cell paste and discard media
172
cell lysis
break open cells using sonication, french press, or freeze/thaw
form homogenate
centrifugation
173
salting out
solubility separation
proteins precipitate out at high salt concentration
salt concentration needed varies with protein solubility
competition between salt ions and protein for interaction with H2O
use salt to precipitate out unwanted proteins then concentrate your protein
use ammonium sulfate
174
why use ammonium sulfate
has a high solubility and low level of interference with proteins
ions from salt can decrease proteins solubility without denaturing it
175
column chromatography phases
mobile phase: sample dissolved in gaseous liquid or liquid
| stationary phase: porous solid matrix in column
176
how column chromatography works
interaction of mobile phase (sample) and stationary phase (matrix in column) retards progress through column according to [charge, size, hydrophobicity, etc.]
separates sample into pure substances (proteins)
heavily used separation technique
177
ion exchange chromatography
separate proteins by net charge
salt gradient low to high
reversible replacement of ions
salt gradient used to vary elution times
178
size-exclusion (gel filtration) chromatography
molecular sieve chromatography
separates according to size and shape
matrix=wet, spongy beads with pores of narrow range in size
large molecules can't pass through pores so they elute first
small molecules slowed by passing through pores
179
exclusion limit
lowest mass not to fit into pore
180
goal of gel chromatography
separate proteins in their full quarternary structures
| molecular masses of unknown substance can be estimated from its position on the plot
181
gel filtration in de-salting
often used for protein
salt retarded in pores
large protein elutes first
182
gel filtration in buffer exchange
pre-equilibrate with desired buffer
| macromolecules will flow fast and elute with equilibrated buffer
183
gel filtration in concentrating sample
add dry gel particles
| imbibe water but not macromolecules
184
gel filtration in other separations
small molecule inhibitors or coenzymes
DNA from protein
large contaminants
products of synthesis from reactants
185
affinity chromatography
specific molecules bound tightly (not covalently)
exploits more specific biochemical property of ligand bonding
eluted with chemical mimic or change in conditions
most powerful chromatographic technique for separation
fusion protein with 6x His tag
elute with imidazole
1 step high yield purification
genetic engineering (plasmid containing affinity tag)
186
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
protein detection method
detergent: denatures protein, binds tightly, masks charge-- acts as negative
polyacrylamide acts as gel matrix and separates by size (MW)
proteins of known size used as markers
187
isoelectric focusing
```
pI=pH at which protein net charge is 0
no SDS--proteins much have charge to migrate
electrophoresis in gel with pH gradient
protein stops migration at pH=pI
separate proteins according to pI
```
188
immunoblotting/ "western blot"
gel electrophoresis on sample containing protein of interest
blot the proteins from the gel onto nitrocellulose
block unoccupied binding sites on nitrocellulose with casein
incubate with rabbit antibody to protein of interest
wash and incubate with enzyme-linked goat anti-rabbit antibody
assay linked enzyme with colorimetric reaction
189
enzyme-linked immunosorbent assay (ELISA)
for small amounts of protein
| large number of samples
190
enzyme assays
measure activity of enzyme
formation of product of reaction
disappearance of reactant (substrate)
191
enzymes
rate enhancement of 10^6-10^17
192
carbonic anhydrase function
facilitates transport of CO2 from tissues where it is produced to the lungs where it is exhaled
193
enzymes as catalysts
mild conditions (25-40 degrees C, 1 atm P, neutral pH)
specificity (stereospecific)
regulation (genetic, allosteric effects--inhibitors, activators, isozymes)
versatility (wide variety of chemical reactions)
194
enzyme specificity
geometrical complementarity
electronic complementarity
lock and key (enzyme and substrate fit together)
induced fit (full complement only in transition state)
enzymes specific in binding chiral substrates and catalyzing their reactions
195
non-covalent forces between E and S
electrostatic
hydrogen bonding
van der Waals
hydrophobic interactions
196
enzyme active site
3D crevice created by AAs from different parts of the primary structure
active site constitutes small portion of enzyme volume
active site creates unique microenvironments
interaction of enzyme and substrate at active site involves multiple weak interactions
enzyme specificity depends on molecular architecture at active site
197
active site residues
can be distant from each other on polypeptide chain (primary structure)
198
cofactors and coenzymes
most common in enzymes catalyzing oxidation/reduction reactions and group transfer
199
cofactors
(metal ions)
| minerals--Zn2+ and coenzymes--NAD+ in YADH)
200
coenzymes
```
co-substrates (transient association)
prosthetic groups (permanent/covalent association--heme)
```
201
holoenzyme
catalytically active enzyme-cofactor complex
202
apoenzyme
no cofactor
| inactive enzyme
203
oxidoreductases
oxidation reduction reactions (electron transfer)
| glyceraldehyde 3-phosphate dehydrogenase
204
transferases
transfer functional groups between molecules
| aminotransferases
205
hydrolases
cleaves molecules by the addition of water
| trypsin
206
lyases
adds atoms or functional groups to a double bond or removes them to form a double bond
fumarase
207
isomerases
remove functional groups within a molecule
| triose phosphate isomerase
208
ligases
join 2 molecules at the expense of ATP hydrolysis
| DNA ligase
209
enzyme kinetics
study of rates of enzyme-catalyzed reactions
210
factors that affect rate
```
concentrations of enzyme
concentrations of ligands (substrate, products, and effectors)
temperature
pH
ionic strength
```
211
what can be determined by enzyme kinetic assay
order of addition of substrates and release of products
enzyme/substrate and enzyme/product complexes formed
affinities of substrate and/or inhibitor for enzyme
kinetic constants, max rate, and specificity
insight into usual intracellular concentrations of substrates and products
physiological direction of reaction (in vivo)
info on identities of AA at active site (pH variation)
info on active site architecture
212
gibbs free energy
thermodynamic property that measures useful energy
213
what 2 things does gibbs free energy measure
1. free energy difference between products and reactants (neg. if spontaneous)
2. free energy of activation required to initiate the conversion of reactants into products (rate)
214
deltaG dependent on
[reactants] and [products]
215
how does enzyme affect gibbs free energy
accelerates attainment of equilibria
| does not shift its position
216
reaction coordinate
minimum free energy pathway
217
transition state
point of highest free energy along coordinate
218
enzymes on transition state
enzymes bind transition state higher than the ground state
219
catalysis in enzyme-catalyzed reactions
results from stabilization of transition state
220
transition state theory assumption
activated complex is in rapid-equilibrium with the reactants which allows thermodynamics of reaction rates
221
larger gibbs free energy of transition state
slower reaction
222
catalysts act by
lowering activation barrier for reaction being catalyzed
223
mechanistic description
elementary reactions comprising overall reactionr
224
rate proportional to
frequency with which reactants collide
225
rate equation
k[A]^a[B]^b
| k is proportionality constant or rate constant
226
reaction order
```
moleclarity of reaction
# of reactants whose concentration dictates rate
```
227
first order
A-->P
| M/s
228
second order
2A-->P
A + B --> P
1/Ms
229
hallmark of first order reaction
time for half of reaction to decompose (half-life) is a constant
230
hallmarks of second order reaction
dependent on initial [A]
231
rate laws for enzyme reactions
E + S = ES = E + P
| when [S] is high converts all E to ES form so 2nd step is "rate-limiting"
232
overall rate of ES production=
sum of reactions for appearance and disappearance
233
assumption of steady state
substrate in great excess
after transient phase [ES] remains constant until substrate is nearly exhausted
[ES] maintains a steady state
234
physical steps of chemical kinetics
binding of substrate to enzyme
structural rearrangements of protein
catalysis (chemical step)
desorption of product
235
michaelis menten equation
Vo= (Vmax[S])/(Km + [S])
236
Kcat
maximum rate of turnover at saturating substrate
lower limit on any intrinsic rate constant
(Vmax/Et)
237
Km
michaelis constant
substrate half concentration at which the reaction velocity is half maximal
(Vmax/2)
238
Kcat/Km
apparent second order rate constant for substrate binding
specificity constant
lower limit of substrate binding rate
239
lineweaver-burke plots
estimates of kinetic constants obtained graphically
| can give non-uniform weighting of points at low substrate concentration
240
Kd
dissociation constant
241
haldane relationship
kinetic parameters of reversible enzyme reaction are not independent of one another
related by equilibrium constant Keq
242
inhibitors
reduce enzyme activity by binding to enzyme and affecting substrate binding or turnover number
243
what do inhibitors mimic
```
substrate
transition state (bind tightest)
```
244
competitive inhibition
```
does not affect Kcat
competitive inhibitor sompetes with substrate for binding site
reduces free [E] available
lines intersect on y-axis
Vo=(Vmax[S])/(aKm + [S])
```
245
uncompetitive inhibition
```
inhibitor binds directly to ES complex (not to free E)
affects Kcat and Km
parallel lines
most often in multi-substrate enzymes
Vo=(Vmax[S])/(Km + a'[S])
```
246
mixed (non-competitive) inhibition
inhibitor bind both E and ES
lines cross left of y-axis
affects Kcat and Km
Vo=(Vmax[S])/(aKm + a'[S])
247
enzyme effects on pH
```
enzymes have bell shape dependence on pH
seen in:
substrate binding
catalytic activity
ionization of substrate
variation in protein structure
```
248
initial velocity experiment at varied pH
provides info on ionizable residues essential for activity
249
bisubstrate enzyme kinetic mechanisms
60% of known biochemical reactions
A, B, C, D=substrates in order they add to enzyme
P, Q, R, S=order they are released from enzyme
E, F, G=stable, different enzyme forms
uni, bi, ter, quad=# of substrates/products
250
ordered bi bi
all reactants must bind before a product is released
A first then B second
lines meet at 1 point on neg x-axis on graph
251
random bi bi
A & B must bind before P or Q released
| A or B can add first
252
ping pong bi bi
one or more product released before all substrates have added
parallel lines on graph
