Enzymes Flashcards
1
Q
Serum ; Reagent
A
Enzyme concentration ; Substrate concentration
2
Q
↑ Enzyme concentration =
Enzyme > substrate, substrate =
A
↑ reaction rate
↑ reaction rate
3
Q
When substrate concentration reaches a maximal value, higher concentration of substrate no longer results in increased rate of reaction
A
Saturation kinetics
4
Q
Nonprotein entities
A
Cofactors
5
Q
Organic compound (Ex. NADP)
A
Coenzymes
6
Q
↑Coenzyme =
A
↑ Velocity
7
Q
Inorganic ions
A
Activators
8
Q
Alters spatial configuration of the enzyme for proper substrate binding
A
Activators
9
Q
Ex. Ca2+ (#1 activator), Zn2+ (LDH), Cl- (AMS), Mg2+ (CK, ALP)
A
Activators
10
Q
Inorganic ion attached to a molecule
A
Metalloenzymes
11
Q
Ex. Catalase, cytochrome oxidase
A
Metalloenzymes
12
Q
Interferes with the enzymatic reactions
A
Inhibitors
13
Q
Binds to the active site of an enzyme
A
Competitive inhibitor
14
Q
Reversible (Substrate > Inhibitor)
A
Competitive inhibitor
15
Q
Binds to the allosteric site (cofactor site)
A
Noncompetitive inhibitor
16
Q
Irreversible
A
Noncompetitive inhibitor
17
Q
Binds to the enzyme-substrate complex
A
Uncompetitive inhibitor
18
Q
↑ Substrate =
A
↑ ES = ↑ Inhibition
19
Q
Same catalytic reactions but slightly different molecular structures ; Fractionation
A
Isoenzymes
20
Q
Ex. NADP
A
Coenzymes
21
Q
= optimum temperature for enzyme activity
A
37’C
22
Q
↑ Temperature =
A
↑ Reaction rate (↑ movement of molecules)
23
Q
Denaturation of enzymes
A
40-50’C
24
Q
Inactivation of enzymes
A
60-65’C
25
For every 10OC increase in temperature, there will be a two-fold increase in enzyme activity
Temperature coefficient (Q10)
26
Most physiologic reactions occur in the pH range of
7-8
27
Storage
Enzymes: = for longer period of time
Substrate and Coenzymes:
LDH (LD4 & 5):
-20’C
2-8’C
Room temperature
28
Mostly increases enzyme concentration
Hemolysis
29
Decreases enzyme concentration
Lactescence or milky specimen
30
Enzyme nomenclature
1st digit:
2nd and 3rd digits:
4th digit(s):
classification
subclass
serial number
31
Enzyme classification
“OTHLIL”
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
32
Redox reaction
Oxidoreductases
33
Dehydrogenases
-Cytochrome oxidase
-LDH
-MDH
-Isocitrate dehydrogenase
-G-6-PD
34
Transfer of a chemical group other than hydrogen from 1 substrate to another
Transferases
35
Kinases, Transaminases, Aminotransferases:
-CK
-GGT
-AST
-ALT
-OCT
36
Hydrolysis/splitting by addition of water
Hydrolases
37
Esterases
-ACP
-ALP
-CHS
-LPS
38
Peptidases
-Trypsin
-Pepsin
-LAP
39
Glycosidases
-AMS
-Galactosidases
40
Removal of groups w/o hydrolysis (product contains double bonds)
Lyases
41
Decarboxylases
-Glutamate decarboxylase
-Pyruvate decarboxylase
-Tryptophan decarboxylase
42
Intramolecular arrangements
Isomerases
43
Glucose phosphate isomerase
Isomerases
44
Ribose phosphate isomerase
Isomerases
45
Joining of 2 substrate molecules
Ligases
46
Synthases
Ligases
47
Water-free cavity
Active site
48
Where the substrate interacts
Active site
49
Cavity other than the active site
Allosteric site
50
May bind regulatory molecules
Allosteric site
51
Coenzyme that is bound tightly to the enzyme
Prosthetic group
52
Apoenzyme + Prosthetic group
Holoenzyme
53
Inactive form of enzyme
Zymogen/proenzyme
54
Shape of the key (substrate) must fit into the lock (enzyme)
Emil Fisher’s/Lock and Key theory
55
Based on the substrate binding to the active site of the enzyme
Kochland’s/Induced fit theory
56
Acceptable theory
Kochland’s/Induced fit theory
57
Enzymes catalyze reactions by lowering the activation energy level that the substrate must reach for the reaction to occur
Enzyme kinetics
58
Enzyme combines w/ only 1 substrate and catalyzes only 1 reaction
Absolute specificity
59
Enzymes combine w/ all the substrates in a chemical group
Group specificity
60
Enzymes reacting w/ specific chemical bonds
Bond specificity
61
Reaction rate depends only on enzyme concentration Independent on substrate concentration
Zero-order reaction
62
Reaction rate is directly proportional to substrate concentration
First-order reaction
63
Independent on enzyme concentration
First-order reaction
64
Measurement of enzyme activity
Change in substrate concentration
Change in product concentration
Change in coenzyme concentration
65
1 micromole of substrate/minute
International Unit
66
Unit 1 mole of substrate/second
Katal
67
Absorbance is made at 10-second intervals for 100 seconds
Nonkinetic assay
68
pH = 10.5 ; 405nm
Alkaline Phosphatase
69
Electrophoresis: (+) Liver Bone (Regan) Placenta Intestine (-)
Alkaline Phosphatase
70
Heat fractionation: (Δ Stable) Regan Placenta Intestine Liver Bone (Δ Labile)
Alkaline Phosphatase
71
Inhibits Regan, placental and intestinal ALP
Phenylalanine
72
Inhibits Nagao ALP
L-leucine
73
Inhibits liver and bone ALP
Levamisole
74
Inhibits bone ALP Methods (ALP)
3M urea
75
= Increased ALP
Low temperature
76
Methods (ALP)
1. Bowers and McComb (PNPP) – IFCC recommended
2. Bessy, Lowry and Brock (PNPP)
3. Bodansky, Shinowara, Jones, Reinhart = BGP (beta glycerophosphate)
4. King and Armstrong = PP (phenylphosphate)
5. Klein, Babson & Read = Buffered PPP (phenolphthalein phosphate)
6. Huggins and Talalay = PPDP (phenolphthalein diphosphate)
7. Moss = ANP (alpha naphthol phosphate)
77
– IFCC recommended
1. Bowers and McComb
78
(PNPP)
1. Bowers and McComb
2. Bessy, Lowry and Brock
2. Shinowara
79
= BGP (beta glycerophosphate)
3. Bodansky, Shinowara, Jones, Reinhart
80
= PP (phenylphosphate)
4. King and Armstrong
1. Gutman and Gutman
81
= Buffered PPP (phenolphthalein phosphate)
5. Klein, Babson & Read
82
= PPDP (phenolphthalein diphosphate)
6. Huggins and Talalay
83
= ANP (alpha naphthol phosphate)
7. Moss
84
Sprue, Hyperparathyroidism, Rickets (children) and osteomalacia (adults)
Increased ALP
85
pH = 5.5 ; 405nm
Acid Phosphatase
86
Sources: Prostate (major), RBC, platelets, bone
Acid Phosphatase
87
pH 7.5 ; 340nm
Aspartate Aminotransferase (AST/SGOT)
88
Sources: Cardiac tissue > Liver > Skeletal muscle > Kidney, pancreas, RBCs
Aspartate Aminotransferase (AST/SGOT)
89
pH 7.5 ; 340nm
Alanine Aminotransferase (ALT/SGPT)
90
Major Source: Liver
Alanine Aminotransferase (ALT/SGPT)
91
Inhibited by L-tartrate ions
Prostatic ACP
92
Inhibited by cupric and formaldehyde ions
RBC ACP
93
Room temperature (1-2 hrs) =
decreased ACP
94
= specific substrate, substrate of choice (endpoint)
Thymolphthalein monophosphate
95
= preferred for continuous monitoring methods
Alpha-naphthyl phosphate
96
Methods (ACP)
1. Gutman and Gutman = PP
2. Shinowara = PNPP
97
= ANP (continuous monitoring)
3. Babsonm Read and Phillips
98
= Thymolphthalein monophosphate (endpoint)
4. Roy and Hillman
99
Methods (AST and ALT)
1. Karmen method = Kinetic
2. Reitman and Frankel = Endpoint
100
Reitman and Frankel
-Color developer:
-Color intensifier:
DNPH
0.4N NaOH
101
Increased Transaminases DeRitis ratio (ALT:AST) >1.0 =
Acute hepatitis (Highest)
102
Increased Transaminases ↑ 20x =
viral or toxic hepatitis
103
Increased Transaminases Moderate elevation =
chronic hepatitis, hepatic cancer, IM
104
Increased Transaminases Slight elevation =
Hepatic cirrhosis, alcoholic hepatitis, obstructive jaundice
105
Smallest enzyme (appears in urine)
Amylase
106
Earliest pancreatic marker
Amylase
107
: most predominant pancreatic AMS isoenzyme in AP
P3
108
Amylase Isoenzymes:
S-type (ptyalin): anodal
P-type (amylopsin): cathodal
109
Samples w/ high activity of AMS should be diluted w/ [?] to prev. inactivation
NaCl
110
= inhibited by wheat germ lectin
Salivary AMS
111
AMS Substrate:
Starch
112
Reducing sugars produced
Saccharogenic
113
Classic reference method (SU)
Saccharogenic
114
Degradation of starch
Amyloclastic
115
Increase in color intensity
Chromogenic
116
Continuous-monitoring technique
Coupled-enzyme
117
Late marker (AP)
Lipase
118
Most specific pancreatic marker
Lipase
119
LPS Substrate:
Olive oil/Triolein
120
Methods (LPS)
1. Cherry Crandal (Reference method)
2. Tietz and Fiereck
3. Peroxidase coupling (most commonly used method)
121
LPS
(Reference method)
(most commonly used method)
1. Cherry Crandal
3. Peroxidase coupling
122
Lacks specificity
Lactate dehydrogenase
123
RBC: 150x than in serum
Lactate dehydrogenase
124
Lactate dehydrogenase Sources:
LD1 (α-HBD) and LD2 = Heart, RBC, Kidneys
LD3 = pancreas, lungs, spleen
LD4 an LD5 = liver and muscle
LD6 = alcohol dehydrogenase
125
= Heart, RBC, Kidneys
LD1 (α-HBD) and LD2
126
= pancreas, lungs, spleen
LD3
127
= liver and muscle
LD4 an LD5
128
= alcohol dehydrogenase
LD6
129
Methods (LDH)
1. Wacker method (forward/direct)
2. Wrobleuski LaDue (reverse/indirect)
3. Wrobleuski Cabaud
4. Berger Broida
130
= pH 8.8, 340 nm, most commonly used
1. Wacker method (forward/direct)
131
= pH 7.2, 2x faster
2. Wrobleuski LaDue (reverse/indirect)
132
10-fold increase (LDH)
Hepatic carcinoma and toxic hepatitis
133
2-3x URL
Viral hepatitis and cirrhosis
134
Creatine Kinase Isoenzymes:
CK-BB
CK-MB
CK-MM
135
= most anodal, brain
CK-BB
136
= myocardium (20%)
CK-MB
137
= least anodal, skeletal and smooth muscles (Major, 94-100%)
CK-MM
138
Total CK: 50x URL (highest)
Duchenne’s muscular dystrophy
139
Most specific indicator of myocardial damage (AMI)
CK-MB
140
Not elevated in angina
CK-MB
141
Methods (CK)
1. Tanzer-Gilbarg (forward/direct) = pH 9.0, 340nm
2. Oliver-Rosalki/ Rosalki & Hess (reverse/indirect) = most commonly used method, faster reaction; pH 6.8, 340nm
142
= pH 9.0, 340nm
1. Tanzer-Gilbarg (forward/direct)
143
= most commonly used method, faster reaction; pH 6.8, 340nm
2. Oliver-Rosalki/ Rosalki & Hess (reverse/indirect)
144
Inside RBCs
Adenylate kinase
145
Interferes w/ CK assay
Adenylate kinase
146
Inhibited by adenosine monophosphate
Adenylate kinase
147
Activate CK
N-acetylcysteine
148
Do not contain CK
Liver cells and RBC
149
Partially restore lost activity of CK
Cleland’s reagent and glutathione
150
Reference method for CK
Electrophoresis
151
CK relative index (CKI) (%) =
CK-MB/Total CK x 100
152
Aldolase Isoenzymes:
Aldolase A = Skeletal muscles
Aldolase B = WBC, liver, kidney
Aldolase C = brain tissue
153
= Skeletal muscles
Aldolase A
154
= WBC, liver, kidney
Aldolase B
155
= brain tissue
Aldolase C
156
Marker for hepatobiliary diseases and infiltrative lesions of the liver
5’ Nucleotidase
157
5’ Nucleotidase Methods:
1. Dixon and Purdon
2. Campbell, Belfield and Goldberg
158
Located in the canaliculi of the hepatic cells
GGT
159
Differentates the source of an elevated ALP level
GGT
160
Sensitive indicator of occult alcoholism
GGT
161
GGT Increased:
Obstructive jaundice
Alcoholic hepatitis (most sensitive)
162
GGT Substrate:
gamma-glutamyl-p-nitroanilide
163
Methods (GGT)
1. Szass 2. Rosalki and Tarrow 3. Orlowski
164
Monitor effects of relaxants (succinylcholine) after surgery
Cholinesterase/ Pseudocholinesterase
165
Marker for organophosphate poisoning (Low CHS)
Cholinesterase/ Pseudocholinesterase
166
Cholinesterase/ Pseudocholinesterase Methods:
1. Ellman technic
2. Potentiometric
167
A.k.a. peptidyldipeptidase A or Kininase II
Angiotensin-Converting Enzyme
168
Converts angiotensin I > angiotensin II (lungs)
Angiotensin-Converting Enzyme
169
Indicator of neuronal dysfunction (Alzheimer’s disease – CSF)
Angiotensin-Converting Enzyme
170
Ferrooxidase enzyme
Ceruloplasmin
171
For hepatobiliary diseases
Ornithine carbamoyl transferase
172
Drug induced hemolytic anemia (primaquine, antimalarial drug)
G-6-PD
