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biochemistry > exam 1 > Flashcards

exam 1 Flashcards

1
Q

water

A

life’s solvent
bent geometry
large electronegative difference
highly polar bc of large dipole moment

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2
Q

hydrogen bonds

A

formed between water molecules to produce highly ordered and open structure
weak interaction

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3
Q

surface tension

A

measure of internal cohesion
force needed to increase surface area
high surface tension in water

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4
Q

coulomb’s law

A

quantifies interaction of ions in solution

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5
Q

dielectric constant

A

measure of solvent’s ability to keep charges apart

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6
Q

water as a solvent

A 
ionization
solvation (hydration)
all interactions
interferes with coulombic forces between ions
high D (charges kept apart)
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7
Q

hydrophobic effect

A

nonpolar molecules do not dissolve in water (hydrophobic, amphiphilic, or amphipathic)
ex. separation of oil and water

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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

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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

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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

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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

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12
Q

is a higher or lower D value a better nonpolar solvent

A

lower

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13
Q

amphiphilic and amphiphatic

A

same thing

molecule that contains both hydrophobic and hydrophilic groups

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14
Q

how do insects walk on water

A

surface tension??

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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???

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16
Q

would a globular protein fold correctly in a solvent of low dielectric constant

A

???

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17
Q

how are all weak interactions fundamentally electrostatic interactions

A

???

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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

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19
Q

order of bond strength

A

???

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20
Q

bronsted acid

A

h+ donor

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21
Q

bronsted base

A

h+ acceptor

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22
Q

acid base reactions

A

concentration of water is essentially unchanged
kw=ion product constant of water
ion concentrations are reciprocally related

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23
Q

the strength of an acid is determined by what

A

dissociation constant (Ka)

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24
Q

Ka

A

dissociation constant

larger Ka=stronger acid

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25
Q

strong acids

A

transfer all protons to h2o

complete ionization

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26
Q

weak acids

A

little ionization

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27
Q

henderson-hasselbalch equation

A

pH= pKa + log([A-]/[HA])

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28
Q

buffers

A

mitigate pH change

consist of acid base conjugate pairs

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29
Q

pK

A

midpoint

pH at which acid and conjugae base are in equal amounts

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30
Q

buffering in blood

A

citric acid cycle produces CO2 as metabolite

carbonic acid readily dissociates into conjugate acid base pair and acts as blood buffer

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31
Q

polyprotic acids

A

multiple pKs
one for each ionization step
steep equivalence points are pH jumps

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32
Q

is a solution with [H+]=10^-4 acidic or basic

A

acidic

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33
Q

how many pK values will be present in a titration of H3PO4

A

4

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34
Q

amino acid stereochemistry

A

chiral alpha carbon

only L-amino acids found in proteins

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35
Q

what make D amino acids

A

racemases

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36
Q

amino acid properties

A 
varied in:
polarity
acidity/basicity
aromaticity
bulk
conformational flexibility
ability to cross link
ability to hydrogen bond
chemical reactivity
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37
Q

amino acid use outside of proteins

A

metabolites

energy sources

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38
Q

glycine

A

simplest AA
only non chiral AA
often in protein sequence for turns in a structure

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39
Q

hydrophobic AA

A 
alanine
valine
leucine
isoleucine
methionine
proline
phenylalanine
tryptophan
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40
Q

alanine

A

utilized in glucogenesis

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41
Q

valine
leucine
isoleucine

A

branched aliphatic chains

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42
Q

methionine

A

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

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43
Q

proline

A

imino acid
closed chain
similar to glycine–found in turns of proteins
derivative= hydroxyproline (in connective tissue-makes stronger)

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44
Q

phenylalanine

A

alanine with benzene ring attached
necessary for synthesis of catecholamines (dopamine, epinephrine)
lack of Phe catabolism associated with phenylketonuria (PKU) and mental retardation

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45
Q

alanine
isoleucine
phenylalanine

A

branched hydrophobic side chains

lack of polar surface area in side chain

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46
Q

tryptophan

A

used as fluorophore
precursor for seretonin–need trp to cross blood-brain barrier
in turkey and milk

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47
Q

psychedelic drugs

A

illegal hallucinagens of the tryptamine family

mimic and interfere with neurotransmitters

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48
Q

uncharged polar amino acids

A 
serine
threonine
asparagine
glutamine
tyrosine
 cysteine
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49
Q

serine

A

alanine with OH

activation site in enzymes regulated by phosphorylation, acetylation

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50
Q

threonine

A

similar to serine
also a target of phosphorylation in enzymes
acts as regulation site

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51
Q

asparagine

glutamine

A

derivatives of aspartate and glutamate

detox of ammonia, transport to live and kidney for urea synthesis

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52
Q

serine

glutamine

A

increased polar surface area due to O and NH2 groups on the side chains

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53
Q

tyrosine

A

pka=10.46
precursor to dopamine and epinephrine (adrenaline)
derivative tyramine in cheese=pick me up
sometimes used a fluorophore

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54
Q

cysteine

A

pka=8.37

forms disulfide bonds which are important for protein structure, connective tissues, hair

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55
Q

cystine

A

break disulfide bonds, shape, reoxidize

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56
Q

positively charged AA

A

lysine
arginine
histidine

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57
Q

lysine

A

pka=10.54

connective tissue biosynthesis

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58
Q

arginine

A

pka=12.48
immediate precursor to urea
hypoargininemia associated with mental retardation

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59
Q

histidine

A

pka-6.04
precursor to histamine (signal molecule that tiggers allergic reactions)
anti-histamine used to block

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60
Q

negatively charged AA

A

aspartate

glutamate

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61
Q

aspartate

glutamate

A

pka=3.9
pka=4.07
neurotransmitter

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62
Q

aspartate

lysine

A

charged side chains and polarity

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63
Q

essential vs nonessential AA

A

essential: obtain through diet
nonessential: synthesized in body

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64
Q

alpha-carboxylic acids

A

pka=2.2-3.5

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65
Q

alpha-amino groups

A

pka=9.4

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66
Q

pI

A

isoelectric point=1/2(pK1 + pK2)

pH of net neutral charge

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67
Q

acidic AA

A

pI (net neutral charge) is between 2 lower pK’s

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68
Q

basic AA

A

pI (net neutral charge) is between 2 higher pK’s

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69
Q

formation of a polypeptide

A

carboxylic end attaches to amino end (amide bond)
water is released (condensation reaction)
dipeptide bond formed

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70
Q

tripeptide

A

3 bonds

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71
Q

oligopeptide

A

several peptide bonds

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72
Q

poly

A

more than 40 bonds

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73
Q

average AA residue

A

110 Da

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74
Q

polypeptide molecular mass

A

ranges from 40-3700 kDa

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75
Q

characteristics of a peptide bond

A

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)

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76
Q

chemistry of peptide bond

A

40% double bond character
shorter bond (increased rigidity)
trans position
flexible and fold

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77
Q

backbone of peptide chain

A

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

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78
Q

phi and psi

A

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

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79
Q

why is 180 planar arrangement most stable angle in a polypeptide

A

steric interference at increased angle between adjacent residues
interaction between molecular orbitals
(side chain interference)

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80
Q

phi angle

A

angle between nitrogen of amino group and alpha carbon

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81
Q

psi angle

A

angle between alpha carbon and carbon of carboxylic group

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82
Q

are phi or psi angles more constrained

A

phi angles

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83
Q

factors that limit rotation

A

peptide bond planarity
side chain bulk
RESONANCE

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84
Q

residue

A

each amino acid in an oligopeptide

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85
Q

nomenclature of peptide bonds

A

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

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86
Q

what limits cis transformation in peptides

A

steric interference

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87
Q

what is the least constrained angle degree

A

180

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88
Q

laws of thermodynamics

A
  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
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89
Q

thermodynamics

A

describes energy flow

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90
Q

life and reproduction require

A

work

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91
Q

work requires

A

energy

ex: synthesis of biomolecules, maintenance of concentration of gradients, movement of muscles

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92
Q

dynamic steady state

A

what we must maintain

energy must be extracted, stored, and channeled

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93
Q

much of cellular biochemical apparatus devoted to

A

acquisition and utilization of energy

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94
Q

organisms

A 
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)
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95
Q

heat and work of a system

A

heat is absorbed BY the system FROM the surrounding and work is done ON the system BY the surroundings

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96
Q

change in energy equation

A

U=energy

deltaU=q+w (heat + work)

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97
Q

cells

A

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

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98
Q

cellular process of energy

A

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

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99
Q

cellular work

A

chemical synthesis, mechanical work, osmotic and electrical gradients, genetic info transfer

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100
Q

metabolites

A

increased energy

simpler than chemical fuels (CO2, H2O, NH3, HPO4 2-)

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101
Q

info-rich macromolecules

A

decreased entropy
simple compounds polymerize to form these
DNA, proteins

102
Q

enthalpy

A

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
Q

spontaneous process

A

characterized by conversion of order to chaos

104
Q

entropy

A

S
disorder
+ deltaS= increased randomness
organisms are entropy poor and demand energy to create order

105
Q

gibbs free energy

A

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
Q

neg. deltaH and pos. deltaS

A

spontaneous at all temperatures

107
Q

neg. deltaH and neg. deltaS

A

spontaneous only at low temperatures

108
Q

pos. deltaH and pos. deltaS

A

spontaneous only at high temperatures

109
Q

pos. deltaH and neg. deltaS

A

not spontaneous at any temperature

110
Q

equilibrium constant

A

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
Q

how does deltaG related to standard free-energy change

A

deltaG=deltaG°+RTlnKeq
at equilibrium deltaG=0
expresses driving force of reaction

112
Q

central dogma of molecular biology

A

DNA –> RNA –> protein

transcription then translation

113
Q

levels of protein structure

A 
structure leads to function
primary
secondary
tertiary
quarternary
114
Q

primary

A

unfolded amino acid sequence (denatured)

115
Q

how long are most proteins

A

50-300 AA long

116
Q

secondary

A

helix (antiparallel or parallel)

117
Q

tertiary

A

one complete protein chain

118
Q

quarternary

A

4 separate chains of hemoglobin assembled into oligomeric protein

119
Q

alpha helix

A 
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
Q

AA and the alpha helix

A

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
Q

helices notation

A

n sub m
n=residues/turn
m=# of atoms in ring enclosed by by H bond

122
Q

beta sheets

A

antiparallel (strands run in opposite directions)–hairpin

parallel (strands run in the same direction)–crossover

123
Q

left handed beta helix

A

tandem repeated sequence

124
Q

fibrous proteins

A

secondary structure dominant
keratin and collagen are common fibrous proteins
2 stranded coiled-coil superhelix

125
Q

keratin

A 
extended coiled-coil structure
hair, horns, nails, feathers, claws
chemically unreactive
macrofibrils
coiled coil forms bc of 7 residue pseudorepeat in primary structure
126
Q

superfamily “coiled-coil proteins”

A

myosin

topomyosin

127
Q

macrofibril

A

tiny part of hair cell

128
Q

collagen

A

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
Q

helix of collagen

A

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
Q

globular proteins tertiary structure

A

3D fold of polypeptide chain
secondary structure spatial arrangement of AA residues far apart in sequence held together by forces including disulfide bonds

131
Q

globular proteins

A

enzymes
transport and receptor proteins (perform chemistry in cell)
more intricate 3D structure

132
Q

tertiary structure domains

A

structurally independent units of single polypeptide chain (sometimes separate function)

133
Q

tertiary structure motif

A

common grouping of secondary structural elements

134
Q

scientist that discovered alpha helix secondary structure of proteins

A

linus pauling

135
Q

interior of globular proteins

A

hydrophilic residues

136
Q

exterior of globular proteins

A

hydrophobic residues

137
Q

protein folding directed by

A

AA interactions

138
Q

how to proteins fold

A

spontaneously into native conformations under physiological conditions
folding is not random–directed by AA
structure stabilized by disulfide bonds
dialyze out denaturing agents

139
Q

beta-mercaptoethanol

A

reduces disulfide bonds

as disulfide bonds are reduced, beta-mercaptoethanol is oxidized and forms dimers

140
Q

assisted disulfide bond formation

A

proper folding can be assisted by enzymes or other proteins

141
Q

disulfide bonds

A

hold unstable state

post-translationally modified proteins–proinsulin–>insulin

142
Q

levinthal’s paradox

A

discrepancy between actual time and calculated time it takes a protein to fold

143
Q

answer to levinthal’s paradox

A

cumulative selection

tendency to retain partly correct intermediates

144
Q

cumulative selection

A

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
Q

what effects protein folding

A

secondary structure

hydrophobic effect

146
Q

secondary structure on protein folding

A 
restricted conformations (helix and sheets)
level of specificity
steric constraints (compact polymers)
short range forces (H bonds, ion pairs, van der Waals)
147
Q

hydrophobic effect

A

mutual exclusion of water

residues predominate in protein interior

148
Q

hierarchical organization

A

polypeptide chains form locally compact structures that associate with similar adjacent structures to form larger compact structures

149
Q

folding funnel model of protein folding

A

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
Q

random conformational search model of protein folding

A

random conformational search=flat surface with narrow hole

NOT CORRECT

151
Q

classic folding model of protein folding

A

classic folding landscape=random search until “canyon” found that leads to native search
faster than random, but not fast enough
NOT CORRECT

152
Q

landscape theory of protein folding

A

folding funnel=energy landscape
conformational adjustments to reduce free energy and entropy until native state reached
no local minima or transient energy barriers

153
Q

molecular chaperones

A

heat shock proteins

bind to hydrophobic surfaces (and release) and facilitate folding

154
Q

obstacles to binding

A

solvent exposure

aggregation with other molecules or itself

155
Q

natively unfolded proteins

A

intrinsically unstructured proteins (IUPs)
bind to other molecule then form stable structure
more common in eukaryotes
molecular versatility important in signaling and regulation

156
Q

amino acids not prevalent in natively unfolded proteins

A

V, L, I, M, W, Y (bulky)

157
Q

amino acids prevalent in natively unfolded proteins

A

Q, S, P, E, K, G, A (polar)

158
Q

protein misfolding diseases

A

bovine spongiform encephalopathy
alzheimer
parkinson
huntington

159
Q

protein dynamics

A

atomic fluctuations: vibrations of individual bonds
collective motions: covalently-linked atoms move as units
triggered conformational changes
fluctuations necessary for reactivity

160
Q

lack of structure provides:

A

more freedom to conform to and interact with a variety of proteins

161
Q

assay for protein verification

A

assay for enzyme lactate dehydrogenase based on fact that product of reaction NADH can be detected spectrophotometrically

162
Q

fractionation techniques

A 
solubility
ionic charge
polarity
molecular size
binding specificity
163
Q

clones

A

collection of identical organisms derived from single ancestor

164
Q

recombinant DNA

A

insert DNA for your protein into an autonomously replicating DNA molecule (cloning vector)

165
Q

host organism

A

what replicates DNA

166
Q

cloning vector

A

circular, auto-replicating DNA “plasmid”

cells become loaded with your protein

167
Q

plasmids

A

extrachromosomal genetic element found in a variety of bacteria
has replication origin

168
Q

transformation

A

laboratory technique that transfers plasmids to host bacterial cell (cells treated to be temporarily permeable to small DNA molecules

169
Q

stringent vs. relaxed control in cloning

A

stringent tied to host replication

relaxed can have much higher number of copies

170
Q

restriction endonucleases

A

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
Q

protein over-expression

A

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
Q

cell lysis

A

break open cells using sonication, french press, or freeze/thaw
form homogenate
centrifugation

173
Q

salting out

A

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
Q

why use ammonium sulfate

A

has a high solubility and low level of interference with proteins
ions from salt can decrease proteins solubility without denaturing it

175
Q

column chromatography phases

A

mobile phase: sample dissolved in gaseous liquid or liquid

stationary phase: porous solid matrix in column

176
Q

how column chromatography works

A

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
Q

ion exchange chromatography

A

separate proteins by net charge
salt gradient low to high
reversible replacement of ions
salt gradient used to vary elution times

178
Q

size-exclusion (gel filtration) chromatography

A

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
Q

exclusion limit

A

lowest mass not to fit into pore

180
Q

goal of gel chromatography

A

separate proteins in their full quarternary structures

molecular masses of unknown substance can be estimated from its position on the plot

181
Q

gel filtration in de-salting

A

often used for protein
salt retarded in pores
large protein elutes first

182
Q

gel filtration in buffer exchange

A

pre-equilibrate with desired buffer

macromolecules will flow fast and elute with equilibrated buffer

183
Q

gel filtration in concentrating sample

A

add dry gel particles

imbibe water but not macromolecules

184
Q

gel filtration in other separations

A

small molecule inhibitors or coenzymes
DNA from protein
large contaminants
products of synthesis from reactants

185
Q

affinity chromatography

A

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
Q

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

A

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
Q

isoelectric focusing

A 
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
Q

immunoblotting/ “western blot”

A

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
Q

enzyme-linked immunosorbent assay (ELISA)

A

for small amounts of protein

large number of samples

190
Q

enzyme assays

A

measure activity of enzyme
formation of product of reaction
disappearance of reactant (substrate)

191
Q

enzymes

A

rate enhancement of 10^6-10^17

192
Q

carbonic anhydrase function

A

facilitates transport of CO2 from tissues where it is produced to the lungs where it is exhaled

193
Q

enzymes as catalysts

A

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
Q

enzyme specificity

A

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
Q

non-covalent forces between E and S

A

electrostatic
hydrogen bonding
van der Waals
hydrophobic interactions

196
Q

enzyme active site

A

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
Q

active site residues

A

can be distant from each other on polypeptide chain (primary structure)

198
Q

cofactors and coenzymes

A

most common in enzymes catalyzing oxidation/reduction reactions and group transfer

199
Q

cofactors

A

(metal ions)

minerals–Zn2+ and coenzymes–NAD+ in YADH)

200
Q

coenzymes

A 
co-substrates (transient association)
prosthetic groups (permanent/covalent association--heme)
201
Q

holoenzyme

A

catalytically active enzyme-cofactor complex

202
Q

apoenzyme

A

no cofactor

inactive enzyme

203
Q

oxidoreductases

A

oxidation reduction reactions (electron transfer)

glyceraldehyde 3-phosphate dehydrogenase

204
Q

transferases

A

transfer functional groups between molecules

aminotransferases

205
Q

hydrolases

A

cleaves molecules by the addition of water

trypsin

206
Q

lyases

A

adds atoms or functional groups to a double bond or removes them to form a double bond
fumarase

207
Q

isomerases

A

remove functional groups within a molecule

triose phosphate isomerase

208
Q

ligases

A

join 2 molecules at the expense of ATP hydrolysis

DNA ligase

209
Q

enzyme kinetics

A

study of rates of enzyme-catalyzed reactions

210
Q

factors that affect rate

A 
concentrations of enzyme
concentrations of ligands (substrate, products, and effectors)
temperature
pH
ionic strength
211
Q

what can be determined by enzyme kinetic assay

A

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
Q

gibbs free energy

A

thermodynamic property that measures useful energy

213
Q

what 2 things does gibbs free energy measure

A
  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
Q

deltaG dependent on

A

[reactants] and [products]

215
Q

how does enzyme affect gibbs free energy

A

accelerates attainment of equilibria

does not shift its position

216
Q

reaction coordinate

A

minimum free energy pathway

217
Q

transition state

A

point of highest free energy along coordinate

218
Q

enzymes on transition state

A

enzymes bind transition state higher than the ground state

219
Q

catalysis in enzyme-catalyzed reactions

A

results from stabilization of transition state

220
Q

transition state theory assumption

A

activated complex is in rapid-equilibrium with the reactants which allows thermodynamics of reaction rates

221
Q

larger gibbs free energy of transition state

A

slower reaction

222
Q

catalysts act by

A

lowering activation barrier for reaction being catalyzed

223
Q

mechanistic description

A

elementary reactions comprising overall reactionr

224
Q

rate proportional to

A

frequency with which reactants collide

225
Q

rate equation

A

k[A]^a[B]^b

k is proportionality constant or rate constant

226
Q

reaction order

A 
moleclarity of reaction
# of reactants whose concentration dictates rate
227
Q

first order

A

A–>P

M/s

228
Q

second order

A

2A–>P
A + B –> P
1/Ms

229
Q

hallmark of first order reaction

A

time for half of reaction to decompose (half-life) is a constant

230
Q

hallmarks of second order reaction

A

dependent on initial [A]

231
Q

rate laws for enzyme reactions

A

E + S = ES = E + P

when [S] is high converts all E to ES form so 2nd step is “rate-limiting”

232
Q

overall rate of ES production=

A

sum of reactions for appearance and disappearance

233
Q

assumption of steady state

A

substrate in great excess
after transient phase [ES] remains constant until substrate is nearly exhausted
[ES] maintains a steady state

234
Q

physical steps of chemical kinetics

A

binding of substrate to enzyme
structural rearrangements of protein
catalysis (chemical step)
desorption of product

235
Q

michaelis menten equation

A

Vo= (Vmax[S])/(Km + [S])

236
Q

Kcat

A

maximum rate of turnover at saturating substrate
lower limit on any intrinsic rate constant
(Vmax/Et)

237
Q

Km

A

michaelis constant
substrate half concentration at which the reaction velocity is half maximal
(Vmax/2)

238
Q

Kcat/Km

A

apparent second order rate constant for substrate binding
specificity constant
lower limit of substrate binding rate

239
Q

lineweaver-burke plots

A

estimates of kinetic constants obtained graphically

can give non-uniform weighting of points at low substrate concentration

240
Q

Kd

A

dissociation constant

241
Q

haldane relationship

A

kinetic parameters of reversible enzyme reaction are not independent of one another
related by equilibrium constant Keq

242
Q

inhibitors

A

reduce enzyme activity by binding to enzyme and affecting substrate binding or turnover number

243
Q

what do inhibitors mimic

A 
substrate
transition state (bind tightest)
244
Q

competitive inhibition

A 
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
Q

uncompetitive inhibition

A 
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
Q

mixed (non-competitive) inhibition

A

inhibitor bind both E and ES
lines cross left of y-axis
affects Kcat and Km
Vo=(Vmax[S])/(aKm + a’[S])

247
Q

enzyme effects on pH

A 
enzymes have bell shape dependence on pH
seen in:
substrate binding
catalytic activity
ionization of substrate
variation in protein structure
248
Q

initial velocity experiment at varied pH

A

provides info on ionizable residues essential for activity

249
Q

bisubstrate enzyme kinetic mechanisms

A

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
Q

ordered bi bi

A

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
Q

random bi bi

A

A & B must bind before P or Q released

A or B can add first

252
Q

ping pong bi bi

A

one or more product released before all substrates have added
parallel lines on graph

biochemistry (2 decks)

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