1 Enzymes Flashcards
(127 cards)
1
Q
what methods can you use to prove that enzymes are protein in nature?
A
chemical hydrolysis
enzyme hydrolysis
conventional protein assays
x-ray crystallography
2
Q
how does chemical hydrolysis prove that enzymes are protein in nature?
A
enzyme interacts with H+ or OH- (acids/bases); broken down into peptides and amino acids
3
Q
how does enzyme hydrolysis prove that enzymes are protein in nature?
A
enzymes react to proteases/proteolytic enzymes; broken down into peptides and amino acids
4
Q
what are some conventional protein assays and what happens?
A
- Lowry method: protein reacts to Cu ions. aromatic residues are oxidated and the solution turns blue
- ninhydrin method: detects ammonia or primary/secondary amines. turns purple
- Folin-phenol method: reacts with any reducing substance
5
Q
how does x-ray crystallography prove enzymes are protein in nature?
A
shows composition of amino acids linked by peptide bonds
6
Q
what is the term for an functional/active compound that consists of either just an apoenzyme or an apoenzyme & an essential prosthetic group?
A
holoenzyme
7
Q
what’s an apoenzyme?
A
the protein part of the enzyme
8
Q
what’s a prosthetic group?
A
non-protein part. aka co-factor
9
Q
list some enzymes that lack a prosthetic group
A
trypsin, chymotrypsin, pepsin
10
Q
list some enzymes that are comprised of both an apoenzyme and prosthetic group
A
PPO (polyphenol oxidase), xanthine oxidase
11
Q
define enzyme active site
A
location on enzyme where catalysis occurs (substrates to products)
12
Q
what is catalysis? what are the 2 steps?
A
the facilitation of a reaction. bonds may be formed or existing bonds may be broken.
2 steps are: 1) binding of substrate 2) transformation of substrate to product
13
Q
what happens if there is binding of a substrate, but no transformation?
A
not catalysis lol
an example of this is when an inhibitor binds instead of a substrate
14
Q
describe the active site
A
3d entity. shaped like pockets and crevices. small (duh)
15
Q
what’s the significance of enzyme specificity?
A
enables consistent formation of products without the formation of undesirable co-products
16
Q
define absolute specificity and give examples of enzymes with this trait
A
very stringent in what they select
ex) glucose oxidase, glucokinase, catalase
17
Q
define group specificity and give examples of enzymes with this trait
A
sects a group of closely related molecules to transform
ex) hexokinase, lipase, proteases
18
Q
what is stereospecificity (with regard to enzymes)?
A
when a reaction selects one stereoisomer to transform (cis/trans)
19
Q
distinguish between racemases and epimerases
A
racemases catalyze inversion around an achiral carbon with only ONE achiral center
epimerases do that, but with more than one achiral center.
if there are >1 achiral centers, it can still be an enantiomer if and only if all of the centers have been mirrored.
20
Q
why do we purify enzymes?
A
to remove interfering compounds, silly
21
Q
what characteristics of enzymes can be manipulated during purification
A
- size
- solubility
- charge
- selective adsorption
22
Q
what are four size based purification techniques?
A
- dialysis
- ultrafiltration
- centrifugation
- gel filtration
23
Q
what happens during dialysis?
A
particles small enough to pass through the semi-permeable membrane move down concentration gradients until equilibrium is achieved. this means that you can’t isolate 100% of a sample using this method
24
Q
what happens during ultrafiltration?
A
pressure is applied to a membrane. more efficient than dialysis
25
what happens during centrifugation?
the sample has the devil spun out of it. also the larger particles settle at the bottom first
26
what happens during gel filtration?
small resins (tubes) are put in a gel. smaller molecules get stuck in the tubes and filter out after larger ones. (aka bigger comes out first)
27
what are three charge based separation techniques?
- ion exchange chromatography
- Electrophoresis
- isoelectric focusing
28
which separation technique involves the use of charged resins?
Ion exchange chromatography
29
define strong/weak cation/anion exchangers
strong - charges on resins stable over wide pH range
weak - small pH range
cation - attracts cations (negatively charged resin)
anion - attracts anions (positively charged resins)
30
sulfonic acid is a...
strong cation exchanger
31
quaternary ammonium is a...
strong anion exchanger
32
carboxy methyl cellulose is a...
weak cation exchanger
33
diethylaminoethane (DEAE) is a...
weak anion exchanger
34
the technique involving the migration/mobility of charged molecules under the influence of a current in an electric field is called:
Electrophoresis
35
what 2 factors does migration/mobility depend on?
charge (q) - proportional
| size (r) - inversely proportional to square of radius
36
what's lauryl sulfate do?
imparts equalizing large net charge on different molecules. after treatment, electrophoresis, they will separate based on size.
37
the technique in which molecules migrate through a pH gradient until they reach their isoelectric point is:
isoelectric focusing (or electric focusing)
38
what's chromatographic focusing?
molecules move through pH gradient until they reach their isoelectric point, but within a column
39
larger enzymes are (more/less) soluble than smaller ones?
less
40
molecules with hydrophobic chains are (more/less) soluble in aqueous solvents
less
41
what's significant about the amino acids: phenylalanine, valine, leucine, and isoleucine?
they all contain hydrophobic side chains
42
molecules with hydrophilic side chains are (more/less) soluble in aqueous solvents
more
43
what's significant about the amino acids: serine, glutamic acid, aspartic acid, and threonine?
they all contain hydrophilic side chains
44
what is salting in?
increasing the ionic strength of a solution increases the solubility of a solute (lower concentrations of salt)
45
what is salting out?
high concentrations of salt, the solubility of the proteins drop sharply and proteins can precipitate out
46
what are three separation techniques based on solubility differences?
- isoelectric precipitation
- salt fractionation
- solvent precipitation
47
the technique involving bringing a solution to a protein's isoelectric point to decrease its solubility and make it precipitate:
isoelectric precipitation
48
what's salt fractionation? why does it work?
the very same thing as salting out babeyy
imparts charge onto protein and limits water available to dissolve it
49
the technique involving the use of organic solvents to precipitate proteins is:
solvent precipitation
50
how does solvent precipitation work?
the solvent strips the thin film of moisture normally present on the enzyme and disrupts the enzyme's activities
do this at 4°C and neutral pH to avoid inactivation.
51
what's a good salt to use for separation purposes?
ammonium sulfate
52
what are good solvents for solvent precipitation?
acetone (preferred), ethanol
53
what are 2 binding site based separation techniques?
- affinity chromatography
| - hydrophobic interaction chromatography (HIC)
54
the technique involving the placement of ligands in a column to limit the movement of the enzyme of interest is:
affinity chromatography
55
the technique involving the use of hydrophobic resins on which to stick the hydrophobic enzymes is called
hydrophobic interaction chromatography
56
what are 4 things you can do to test for enzyme purity?
- homogeneity (presence of a single band in electrophoresis)
- chromatography behavior (graph absorbance vs fraction #: pure curve will be bell shaped)
- activity testing
- isoelectric focus
57
name the enzyme groups in order
1) Oxidoreductase
2) Transferase
3) Hydrolase
4) Lyase
5) Isomerase
6) Ligase
58
what group of enzymes catalyze redox reactions? what are some examples?
Group 1 - Oxidoreductases
PPO, GOX (removes sweetness and oxygen from egg white and powdered milk), DOPA, Xanthine
59
what group of enzymes catalzye the transfer of groups from one molecule to another? what are some examples?
Group 2 - transferases
transglutaminase (TGase): transfers groups between gamma-carboxamide residue of glutamine to epsilon-NH2 group of lysine, liberating ammonia and forming an isopeptide bond (C=O)-NH-lys. makes molecules bigger. used to prepare consolidated meats, or to firm up soft fish flesh in surimi.
60
1) what groups get transferred by TGase?
| 2) what molecules does TGase transfer groups between?
1) γ-carboxamide and ε-NH2
| 2) glutamine and lysine respectively
61
what group of enzymes break down larger molecules into smaller ones using WATER as a CO-REACTANT? examples?
Group 3 - Hydrolases
ex) proteolytic enzymes, amylases, lipases
62
what's the most important group of enzymes in the food industry?
group 3
63
what group of enzymes break or create DOUBLE BONDS?
Group 4 - Lyases
ex) fumarase (removes db), histidine decarboxylase
64
what group of enzyme does aldolase belong to?
lyase. it cleaves fructose-1,6-bisphosphate into G3P and DHAP, forming a double bond
65
what group of enzymes converts molecules into isomers? examples?
Group 5 - isomerases
ex) glucose isomerase (converts glucose to fructose)
66
what group of enzymes put smaller molecules together to make larger ones? examples?
group 6 - Ligases
ex) stringing together of nucleotides, carboxylase (formation of oxaloacetate using pyruvate and CO2)
67
by what mechanism does enzyme catalysis occur?
enzymes decrease the activation energy of a reaction.
68
```
ΔG = ?
ΔH¢ = ?
ΔS = ?
R = ?
Ea = ?
```
```
ΔG = change in free energy of activation
ΔH¢ = change in enthalpy
ΔS = change in entropy
R = universal gas constant
Ea = activation energy
```
69
you can increase rate of catalysis by (increasing/decreasing) concentration of enzyme and (increasing/decreasing) concentration of substrate
increase, increase
70
rate Rx depends on what parts of the enzyme reaction?
[E], [S], T, stability of ES, [p], pH
71
how do you measure Rx rates?
- appearance of P
| - disappearance of S
72
what two methods of measurements can you use to determine Rx rate
- initial rate method (take measurements immediately after zero time; true measurement bc no inhibitory products yet)
- end-point method (take measurements after a set amount of time after zero time; no expensive equipment or skill required)
73
what are the 3 stages of enzymatic activity curve?
pre-steady state, steady state, post steady state
74
state where curve appears exponentially shaped
pre-steady state
75
state where curve appears linear
steady state
76
state where curve appears like a horizontal line
post steady steady state
77
what are K1, K2, and K3?
```
K1 = putting together of E and S
K2 = dissociation of ES back to E+S
K3 = formation of E + P from ES
```
78
K1 = K2 + K3
what does this mean?
ES is breaking down at the same rate it is formed
79
in what state are catalytic measurements usually taken?
steady state
80
V0 = ?
initial/observed velocity
1/2 Vmax
81
how do you find vmax?
stick a tangent line approximately where the leveling off of the curve is and then read where it intersects with the y axis
82
Km = ?
ratio of breakdown of complex to formation (K3+K2)/K1 - represents affinity of E for S
*numerically equal to [S] when: V0=1/2Vmax*
83
how useful is the michaelis-menten plot?
not useful in practice because there is no true levelling off of the curve. it actually keeps going up, so you don't get a tru Vmax or Km
instead we use lineweaver-burke method
84
what does the lineweaver-burke plot look like?
linear
85
what is the slope and intercept of the lineweaver-burke plot?
slope: Km'/Vmax
int: 1/Vmax
86
what does Vmax signify?
ease of catalysis
87
what signifies catalytic efficiency?
Vmax/Km' ratio
higher ratios = more economical
88
what do enzyme inhibitors do?
minimize activity/reduce rates of enzyme catalysis
89
what are the 3 types of reversible inhibition?
1) competitive
2) non-competitive
3) uncompetitive
90
when "I" binds to active site, this is ___ inhibition
competitive
91
when "i" binds not at active site and prevents transformation of S to P, this is ___ inhibition
noncompetitive
92
when "i" binds to site on "E" that becomes available only after "S" has bound to active site (i.e. to the ES complex), this is ___ inhibition
uncompetitive
93
benefits of enzymes in food processing?
- viewed as natural
- specific
- don't need high concentrations
- no need for expensive and corrosive equipment
- easy to stop enzyme action with mild heat treatment
- easy removal
94
what enzymes are involved in cheese making?
rennin/rennet, pepsins/chymosins
peptide binds calcium ions, bringing other caseins together to form curd
95
how are proteases used in the beer industry?
if you don't use proteases to break down proteins, then at a low temp you will find sediments at the bottom. the proteases keep peptides in solution. this is called *chill proofing)
96
what use are amylases in baking?
break down starches into simple sugars so that yeast can eat it and release CO2, allowing loaves to swell
97
what use are proteases in baking?
modify proteins like gluten to make the product more pliable
98
what use are lipoxidases in baking?
bleaches flour by breaking down carotenoids
99
what is TGase used for????
slapping together of meat scraps into a larger hunk o'meat
100
what are lipases used for in the meat industry?
flavors
101
what are proteases used for in the meat industry?
texture, solubilization
102
what are the primary reactions in foods catalyzed by enzymes? (4)
- protease
- carbohydrase
- lipase
- oxidoreductase
103
what 2 types of proteases are there/?
1) endoproteases - act randomly
| 2) exoproteases - act at end of protein molecules
104
what subgroups do exoproteases attack?
amino peptidase and carboxy peptidase
105
what are endoproteases used for?
converting proteins from solid to liquid (you want extensive breakdown for small liquified molecules)
106
what are exoproteases used for?
debittering agents. done by removing amino acid from the terminals of proteins
107
generally speaking, action of lipases is (desirable/undesirable). why?
undesirable. FFAs formed are less stable than the acyl glycerides and are responsible for rancid odors
108
when is lipolysis desirable?
characteristic flavors of certain food products like buttermilk, roquefort, milk chocolate, margarines, baked goods, coffee whiteners, imitation dairy products, soups, pizza)
109
what are 7 important group 1 enzymes?
- ascorbic acid oxidase
- PPO
- glucose oxidase (GOX)
- catalase
- lipoxygenase (lipoxidase)
- xanthine oxidase
- peroxidase
110
what properties are enzymes good for modifying?
- solubility
- functional properties
- flavor
111
what is bitterness due to?
hydrophobic amino acids at terminal ends of proteins
112
significance of whey and proteases?
```
whey has a lot of good proteins:
- lactalbumins
- lactoglobulins
- albumin
- immunoglobulin
proteases hydrolyze them into hydrolysates which are less allergenic than the unhydrolyzed versions
```
113
how do can proteases help with Celiac's?
can hydrolyze wheat proteins to break down gluten to form peptides and hydrolysates that can be used for food flavorants
114
how are proteases used in the making of alcoholic beverages?
flavor development
115
how do enzymes contribute to dental care?
plaque is comprised of mouth bacteria, dextrins/sugars and glycoproteins.
- lysozymes break down bacteria cell walls
- dextranases break down sugars
- proteases break down glycoproteins
116
how do enzymes get rid of hair?
hair contains keratin proteins. use keratinase to break it down, but don't leave it too long on the skin or else you can irritate your skin
117
how do enzymes improve tea solubility?
tannins in tea are large, insoluble molecules. *tannases* break them down into them down to make them more soluble in cold teas
118
do you experience more flavor when a beverage is hot or cold?
warm. molecules move faster and interact with taste receptors more
119
how do enzymes help with the preparation of sturgeon roe?
there's a membrane that must be removed. traditionally, eggs are shaken over a screen, but this has high losses.
enzymes increase yields
120
how do enzymes help with the deskinning/descaling of fish
use macerating enzymes to cause less cut damage in fish fillets
121
temperature based control methods
blanching, freezing, chilling, pasteurization
122
water activity based control methods
salting, dehydration
123
chemical based control methods
sulfites, acids, alkalis, antioxidants, chelating agents
124
what are some novel methods of enzymatic control?
- high pressure treatment processing
- enzyme treatment using killer enzymes/anti-enzymes
- inhibitors
- chemical modification
- ionizing radiation
125
how does high pressure treatment work?
causes compaction in molecule. the change in volume is enough to change the configuration of the enzyme and causes a loss in activity
126
what is one thing you can do for chemical modification?
attach alcohols to essential residues
127
how does ionizing radiation work?
causes formation of free radicals that can polymerize causing a reduction in enzyme activity
