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