Enzyme Flashcards
What are Enzymes?
Proteins, Biological catalysts Created in the body Supply energy/chemical changes in the body Muscle contraction Nerve conduction Respiration, reproduction Digestion or nutrient degradation, growth Maintaining body temp
Apoenzyme
heat-sensitive protein portion. Requires a coenzyme
Coenzyme
organic co-factors that resemble vitamins (NAD🡪NADH)
Haloenzyme:
apoenzyme + cofactor
Cofactor
nonprotein molecule necessary for enzyme activity
Metalloenzymes
inorganic cofactor (Cl-, Zn2+, Cu 2+, Ca2+Mg2+)
Zymogen
Inactive Form
Absolute specificity
catalyze 1 specific substrate or reaction
Group specificity
catalyze substrates with similar structural groups
Bond Specificity
catalyzing reaction based upon a certain type of bond
Stereospecificity
Stereoisomer specificity - Catalyze reactions with only certain optical isomers
Oxidoreductases
catalyze an oxidation–reduction reaction between two substrates (LD)
Transferases:
catalyze transfer of a group other than hydrogen from one substrate to another (AST)
Hydrolases
catalyze hydrolysis of various bonds (Amylase)
Lyases
catalyze removal of groups from substrates without hydrolysis; product contains double bonds
Isomerases
catalyze interconversion of geometric, optical, or positional isomers (phosphohexose isomerase) α glucose🡪β glucose
Ligases
catalyze joining of two substrate molecules, coupled with breaking of pyrophosphate bond in ATP
Energy of Activation (EA)
energy required to raise 1 mole of substrate to form the activated complex (IU/L)
Enzymes catalyze reactions by lowering EA level.
Enzyme-Substrate Complex
provides free energy required for the reaction.
Reaction is allowed to proceed without additional energy
↓ energy barrier = ↑ product created
Vmax
substrate concentration high enough that all enzyme is bound to substrate and all active sites are engaged
Michaelis-Menten constant (Km)
substrate concentration in moles/Liter when the speed of enzymatic reaction = ½ Vmax
Represents relationship between reaction speed & substrate concentration
Km is a constant and remains the same for a given enzyme-substrate pair under given conditions
First-order kinetics
The velocity is directly proportional to the substrate concentration
Zero-order kinetics
The reaction rate is independent of substrate concentration
Clinical lab- most common measurements
Unit of Measurement
International Unit (IU) = 1 µmol of product per minute under standard conditions Expressed as U/L (units/liter)
Substrate Concentration
Increases rate of reaction
Zero-order rxn: excess substrate required so that no more than 20% is converted to product in a normal reaction
Enzyme concentration
Higher the enzyme level, the faster the reaction will proceed
More substrate converted to product
pH
Optimal body pH from 7.0 – 8.0
Temperature
↑ temp will ↑ rate of reaction
↓ temp reversibly inactivates enzymes
25oC, 30oC, 37oC
Temperature > 40 - 50oC result in enzyme denaturation
Cofactors
Metallic (Ca2+, Fe2+, Mg2+, Mn2+, K+
Inhibitors
Competitive: physically bind to enzyme active site
Noncompetitive: reversibly binds to a site other than enzyme active site
Uncompetitive: binds to ES complex → [substrate]
Isoenzymes
Multiple forms of an enzyme that can catalyze the enzyme’s characteristic reaction
LD 1: found in rbc; LD5- found in liver
Used to determine origin of disease
Differentiated by: electrophoresis, resistance to heat or chemicals
Measurement of Enzyme Mass
Immunoassay methodologies are used to quantify enzymes
Enzymes as Reagents
Used to measure many non-enzymatic constituents in serum
Used as reagents to quantify analytes that are substrates for corresponding enzyme
Used as reagents in competitive and noncompetitive immunologists (HIV, therapeutic drugs, cancer antigens)
Horseradish perioxidase, ALP, G6PD
Creatine Kinase (CK)
cytoplasmic and mitochondrial enzyme that catalyzes the reversible phosphorylation of creatine by adenosine triphosphate (ATP)
Equilibrium of the CK reaction is dependent on pH
very important in muscle tissue.
Allows high-energy phosphate to be stored in a more stable form than ATP
Dimer with 2 subunits
B (brain) and M (muscle)
Tissue source: (1) muscle (2) brain (3) heart
CK highest in infancy and childhood; decreases as we age
Inverse relationship with thyroid function
Hypothyroidism = ↑CK
CK-2
rises 4-6 hrs post AMI, peaks at 24 hrs and returns to normal within 2-3 days
CK-3
rises with muscular dystrophy (Duchenne type
CK-1
tumor marker for prostate and lung cancer
Childbirth & hypothermia
Hypothyroidism
Specimen Collection CK
Serum is the specimen of choice
Store in dark place; CK is light sensitive
Not affected by hemolysis but adenylate kinase (AK) released by rbcs reacts with ADP- ATP (Oliver-Rosalki) causing increase CK
CK activity is unstable and rapidly lost during storage
4 hrs at room temperature
48 hrs at 4oC
1 month at -20oC
CK Reference Range
Males: 52–236 U/L Females :38–176 U/L Affected by: Age, Physical activity Race, Bed rest (even overnight can decrease CK)
Testing Methods: Oliver-Rosalki
Creatinine phosphate + ADP →┴(𝐶𝐾, 𝑀𝑔2+@ 𝑝ℎ=6.7)Creatine + ATP
ATP + Glucose →┴𝐻𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒 Glucose-6-phosphate + ADP Inhibited by: Ca2+ : fix by adding EDTA or add extra 𝑀𝑔2+ Preferred lab method Measured at 340 nm Optimal pH = 6.8 Other Methods Electrophoresis Ion-exchange chromatography RIA, EIA Immunoinhibition
Lactate Dehydrogenase (LD)
Tissue source
Highest concentration in heart, liver, skeletal muscle
Also found in kidney, erythrocytes
LD is a nonspecific marker
Important enzyme in the Embden-Meyerhoff
Tissue concentration 500 times higher than serum levels
Diagnostic significance LD
pernicious anemia, hemolytic disorders, viral hepatitis, cirrhosis, acute myocardial infarction, pulmonary infarct, skeletal muscle disorders, leukemia
In healthy people the major fractions appear as follows:
LDH-2 ⇾ LDH-1 ⇾ LDH-3 ⇾ LDH-4 ⇾ LDH-5
Heart Attack (AMI)
LDH Flip: when LD-1 > LD-2 = heart attack
Rises 12-24 hrs, peaks 48-72 hrs and returns to normal within 7-14 days after AMI
Two subunits LD
H (heart) and M (muscle) form five isoenzymes.
LD-1 (HHHH)
Heart, RBC and kidney
LD-2 (HHHM)
Heart, RBC and kidney
LD-3 (HHMM)
Spleen, lungs, and many tissues
LD-4 (HMMM)
Liver and skeletal muscle
LD-5 (MMMM)
Liver and skeletal muscle
Specimen Collection LD
Serum, no hemolysis
Stored 72 hrs at room temp= no loss of activity
LD-5 decreased at freezing -20oC
Reference range: LD
L🡪P: 100-224 U/L at 37°C
P🡪L: 80 - 300 U/L at 37°C
Source of error: LD
any degree of hemolysis
Enzyme unstable in serum
Store samples at 25oC rather than 4oC and measure within 24 hrs of collection
LD analysis
Most current methods measure the interconversion of NAD+ to NADH at 340 nm. Wacker procedure Lactate to pyruvate reaction Most often used pH 8.8-9.8 = forward reaction pH 7.4 – 7.8 = reverse reaction
Troponin
Cardiac-specific troponin I (cTnI) Cardiac-specific troponin T (cTnT) Specific for cardiac tissue High diagnostic specificity and sensitivity Released early after AMI Remain elevated for long period of time Very low or undetectable in serum of normal patients Very few interfering substances
Troponin False (+)
Heterophile antibodies
Rheumatoid factor
Troponin False (-)
Bilirubin, hemoglobin
Interfering factor
Circulating cTnI autoantibodies
Natriuretic Peptides (NP)
Regulate fluid volume, blood pressure and electrolyte balance
Cleared and made by the kidneys
Type NP
Atria natriuretic peptide (ANP): cardiac atria
Brain natriuretic peptide (BNP): cardiac ventricles
ANP & BNP released in response to atrial. Ventricular stretching from volume overload, renal and liver disorders
Biomarker for congestive heart failure
↑BNP = severe heart failu
Cytokines
Protein secreted by cells to attract and direct target cells
interleukin-6 (IL-6)
Produced by monocytes & lymphocytes
Acute phase reactant
Elevated in patients with AMI
Leptin
Hormone
Regulates body weight, expressed by adipocytes
Independent risk factor associated with CVD
Low levels associated with unstable coronary artery disease (CAD)
Pregnancy-Associated Plasma Protein A (PAPP-A)
Typically measured to detect Down’s Syndrome
Detects unstable acute coronary syndrome (ACS) without increased concentration of cTn
Placental Growth Factor (PlGF)
Attracts monocytes
Regulates vascular endothelial growth factor (VEGF)
Biomarker for plaque instability, myocardial ischemia, ACS
Aspartate Aminotransferase (AST)
Serum glutamic oxaloacetic transaminase (SGOT or GOT)
Tissue source: AST
cardiac tissue, liver, skeletal muscle
Diagnostic significance: AST
hepatocellular disorders (viral hepatitis, cirrhosis), skeletal muscle disorders (muscular dystrophies, inflammatory conditions), pulmonary embolism
Source of error: AST
Hemolysis
stable at room temp for 48 hrs
3–4 days at refrigerated temperatures
Specimen Collection: AST
serum
Assay: Karmen method AST
2 reactions pH = 7.3 – 7.8 340 nm Malate dehydrogenase is the indicator reaction measuring the decrease in absorbance at 340 nm as NADH is oxidized to NAD+.
Reference range: AST
5–35 U/L (37°C)
Alanine Aminotransferase (ALT)
Glutamic pyruvic transaminase (SGPT or GPT)
More specific than AST for liver disorders
found in Liver tissue
De Ritis ratio (AST/ALT)
Aids in diagnosing liver disease
Normal ratio: 0.7 – 1.4
Alcoholic liver disease/cirrhosis: AST>ALT and is greater than 1
Ratio >2 alcoholic hepatitis or alcoholism
Viral hepatitis, obstructive liver disease and acute inflammatory disease ALT>AST and is less than 1 (ratio < 1
Diagnostic significance ALT
hepatic disorders
Reference range ALT
7 - 45 U/L (37°C)
Specimen Collection ALT
Serum; measure within 24 hrs; decrease activity at 4o and -20°C
ALT stable at -70°C
avoid hemolysis: ALT 5-8 times higher in rbcs than serum
Assay: ALT
Wroblewski and LaDue Method
pH = 7.3 – 7.8
340 nm
Alkaline Phosphatase (ALP)
found in all tissues of the body esp. at or near cell membranes
Highest concentration in hepatocytes
Diagnostic significance: ALP
hepatobiliary (biliary tract obstruction)
bone (Paget’s disease, osteomalacia, rickets, hyperparathyroidism, osteogenic sarcoma) disorders
Separation of isoenzymes led to discovery of abnormal enzyme carcinoplacental(Regan)
Reference range: ALP
42-128 U/L (30°C)
(M/F 20-50 y/o)
Specimen Collection: ALP
Serum or heparin samples Stored at room temp; measure in 4 hrs Levels drop slowly at 4oC (2%/day) Hemolysis acceptable Cannot use any other anticoagulant due to the binding of Mg2+ and Zn2+ to Ca2+
Source of error: ALP
Hemolysis
assays should be run as soon as possible after collection
Enzyme activity ↑ upon standing at RT or 4o
high-fat meal
Group O or B secretors = ↑ ALP
Assay: Bowers & MaComb (ALP)
p-nitrophenyl-phosphate (colorless) is hydrolyzed to p-Nitrophenol (yellow)
405 nm
γ-Glutamyltransferase (GGT)
Present in serum and all cells except muscle
kidney, brain, prostate, pancreas, liver
Diagnostic significance: GGT
hepatobiliary disorders (biliary tract obstruction), hepatic parenchyma, alcoholism,
acute pancreatitis, diabetes mellitus,
myocardial infarction
NOT elevated in bone disease
Used to diagnose drug & alcohol abuse
Used to measure growth during pregnancy and in children
Assay for enzyme activity: GGT
γ-glutamyl-p-nitroanilide is most widely accepted substrate used in GGT analysis
Absorbance @ 405-420 nm
Specimen collection:
Serum
Source of error: GGT
stable for 1 week at 4°C
hemolysis not a concern
Reference range: GGT
male: 6–55 U/L (37°C)
female: 5–38 U/L (37°C)
Amylase (AMS)
acinar cells of pancreas & salivary glands
Diagnostic significance: AMS
Nonspecific finding
acute pancreatitis
disorders causing salivary gland lesions (mumps, parotitis)
intraabdominal diseases
Assay for enzyme activity: AMS
Requires Ca2+ and Cl- for activation Immunoassay Interference: opiates, morphine Serum / urine stable RT for 1 week or 4oC for 2 months
AMY Methodologies
Maltotetraose reaction
Used in many instruments
Most common
AMY → Maltose phosphorylase → β-Phosphoglucose mutase → Glucose-6-phosphate
Reference Range: AMS
Serum: 40-140 U/L
Urine: 24-400 U/L
Lipase (LPS)
primarily in pancreas
also in stomach & small intestine
Diagnostic significance: LPS
acute pancreatitis
other intraabdominal diseases (penetrating duodenal ulcers, perforated peptic ulcers, intestinal obstruction, acute cholecystitis)
Assay for enzyme activity: LPS
turbidimetric (simple and rapid)
Colorimetric (perioxidase or glycerol kinase)
Enzymatic LPS reactions have largely replaced titrimetric and turbidimetric methodologies
Source of error: LPS
stable in serum for 1 week at room temperature & 3 weeks at 4°C; hemolysis
Reference range: LPS
<45 U/L
LPS 🡪 Glycerol kinase 🡪 L-α-glycerophosphate kinase 🡪 Peroxidase
Acid Phosphatase (ACP)
prostate, bone, liver, spleen, kidney, erythrocytes, platelets
Diagnostic significance: ACP
prostatic carcinoma, hyperplasia of prostrate, prostatic surgery,
osteoclasts, Paget’s disease,
breast cancer with bone metastases, Gaucher’s disease
Assay for enzyme activity: ACP
same techniques as in alkaline phosphatase, except performed in an acid pH
Reference range: ACP
prostatic ACP: 0–3.5 ng/mL
Source of error: ACP
Serum should be separated from red cells as soon as blood has clotted; no hemolysis
serum should be used immediately, frozen
acidified to pH = 6.5 stable for 2 days at room temp
