Enzyme

Question 1

What are Enzymes?

Answer 1

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

Question 2

Apoenzyme

Answer 2

heat-sensitive protein portion. Requires a coenzyme

Question 3

Coenzyme

Answer 3

organic co-factors that resemble vitamins (NAD🡪NADH)

Question 4

Haloenzyme:

Answer 4

apoenzyme + cofactor

Question 5

Cofactor

Answer 5

nonprotein molecule necessary for enzyme activity

Question 6

Metalloenzymes

Answer 6

inorganic cofactor (Cl-, Zn2+, Cu 2+, Ca2+Mg2+)

Question 7

Zymogen

Answer 7

Inactive Form

Question 8

Absolute specificity

Answer 8

catalyze 1 specific substrate or reaction

Question 9

Group specificity

Answer 9

catalyze substrates with similar structural groups

Question 10

Bond Specificity

Answer 10

catalyzing reaction based upon a certain type of bond

Question 11

Stereospecificity

Answer 11

Stereoisomer specificity - Catalyze reactions with only certain optical isomers

Question 12

Oxidoreductases

Answer 12

catalyze an oxidation–reduction reaction between two substrates (LD)

Question 13

Transferases:

Answer 13

catalyze transfer of a group other than hydrogen from one substrate to another (AST)

Question 14

Hydrolases

Answer 14

catalyze hydrolysis of various bonds (Amylase)

Question 15

Lyases

Answer 15

catalyze removal of groups from substrates without hydrolysis; product contains double bonds

Question 16

Isomerases

Answer 16

catalyze interconversion of geometric, optical, or positional isomers (phosphohexose isomerase) α glucose🡪β glucose

Question 17

Ligases

Answer 17

catalyze joining of two substrate molecules, coupled with breaking of pyrophosphate bond in ATP

Question 18

Energy of Activation (EA)

Answer 18

energy required to raise 1 mole of substrate to form the activated complex (IU/L)
Enzymes catalyze reactions by lowering EA level.

Question 19

Enzyme-Substrate Complex

Answer 19

provides free energy required for the reaction.
Reaction is allowed to proceed without additional energy
↓ energy barrier = ↑ product created

Question 20

Vmax

Answer 20

substrate concentration high enough that all enzyme is bound to substrate and all active sites are engaged

Question 21

Michaelis-Menten constant (Km)

Answer 21

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

Question 22

First-order kinetics

Answer 22

The velocity is directly proportional to the substrate concentration

Question 23

Zero-order kinetics

Answer 23

The reaction rate is independent of substrate concentration

Clinical lab- most common measurements

Question 24

Unit of Measurement

Answer 24

International Unit (IU) = 1 µmol of product per minute under standard conditions
Expressed as U/L (units/liter)

Question 25

Substrate Concentration

Answer 25

Increases rate of reaction | Zero-order rxn: excess substrate required so that no more than 20% is converted to product in a normal reaction

Question 26

Enzyme concentration

Answer 26

Higher the enzyme level, the faster the reaction will proceed More substrate converted to product

Question 27

pH

Answer 27

Optimal body pH from 7.0 – 8.0

Question 28

Temperature

Answer 28

↑ temp will ↑ rate of reaction ↓ temp reversibly inactivates enzymes 25oC, 30oC, 37oC Temperature > 40 - 50oC result in enzyme denaturation

Question 29

Cofactors

Answer 29

Metallic (Ca2+, Fe2+, Mg2+, Mn2+, K+

Question 30

Inhibitors

Answer 30

Competitive: physically bind to enzyme active site Noncompetitive: reversibly binds to a site other than enzyme active site Uncompetitive: binds to ES complex → [substrate]

Question 31

Isoenzymes

Answer 31

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

Question 32

Measurement of Enzyme Mass

Answer 32

Immunoassay methodologies are used to quantify enzymes

Question 33

Enzymes as Reagents

Answer 33

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

Question 34

Creatine Kinase (CK)

Answer 34

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

Question 35

CK-2

Answer 35

rises 4-6 hrs post AMI, peaks at 24 hrs and returns to normal within 2-3 days

Question 36

CK-3

Answer 36

rises with muscular dystrophy (Duchenne type

Question 37

CK-1

Answer 37

tumor marker for prostate and lung cancer Childbirth & hypothermia Hypothyroidism

Question 38

Specimen Collection CK

Answer 38

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

Question 39

CK Reference Range

Answer 39

``` Males: 52–236 U/L Females :38–176 U/L Affected by: Age, Physical activity Race, Bed rest (even overnight can decrease CK) ```

Question 40

Testing Methods: Oliver-Rosalki

Answer 40

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 ```

Question 41

Lactate Dehydrogenase (LD)

Answer 41

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

Question 42

Diagnostic significance LD

Answer 42

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

Question 43

Heart Attack (AMI)

Answer 43

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

Question 44

Two subunits LD

Answer 44

H (heart) and M (muscle) form five isoenzymes.

Question 45

LD-1 (HHHH)

Answer 45

Heart, RBC and kidney

Question 46

LD-2 (HHHM)

Answer 46

Heart, RBC and kidney

Question 47

LD-3 (HHMM)

Answer 47

Spleen, lungs, and many tissues

Question 48

LD-4 (HMMM)

Answer 48

Liver and skeletal muscle

Question 49

LD-5 (MMMM)

Answer 49

Liver and skeletal muscle

Question 50

Specimen Collection LD

Answer 50

Serum, no hemolysis Stored 72 hrs at room temp= no loss of activity LD-5 decreased at freezing -20oC

Question 51

Reference range: LD

Answer 51

L🡪P: 100-224 U/L at 37°C | P🡪L: 80 - 300 U/L at 37°C

Question 52

Source of error: LD

Answer 52

any degree of hemolysis Enzyme unstable in serum Store samples at 25oC rather than 4oC and measure within 24 hrs of collection

Question 53

LD analysis

Answer 53

``` 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 ```

Question 54

Troponin

Answer 54

``` 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 ```

Question 55

Troponin False (+)

Answer 55

Heterophile antibodies | Rheumatoid factor

Question 56

Troponin False (-)

Answer 56

Bilirubin, hemoglobin Interfering factor Circulating cTnI autoantibodies

Question 57

Natriuretic Peptides (NP)

Answer 57

Regulate fluid volume, blood pressure and electrolyte balance Cleared and made by the kidneys

Question 58

Type NP

Answer 58

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

Question 59

Cytokines

Answer 59

Protein secreted by cells to attract and direct target cells

Question 60

interleukin-6 (IL-6)

Answer 60

Produced by monocytes & lymphocytes Acute phase reactant Elevated in patients with AMI

Question 61

Leptin

Answer 61

Hormone Regulates body weight, expressed by adipocytes Independent risk factor associated with CVD Low levels associated with unstable coronary artery disease (CAD)

Question 62

Pregnancy-Associated Plasma Protein A (PAPP-A)

Answer 62

Typically measured to detect Down’s Syndrome | Detects unstable acute coronary syndrome (ACS) without increased concentration of cTn

Question 63

Placental Growth Factor (PlGF)

Answer 63

Attracts monocytes Regulates vascular endothelial growth factor (VEGF) Biomarker for plaque instability, myocardial ischemia, ACS

Question 64

Aspartate Aminotransferase (AST)

Answer 64

Serum glutamic oxaloacetic transaminase (SGOT or GOT)

Question 65

Tissue source: AST

Answer 65

cardiac tissue, liver, skeletal muscle

Question 66

Diagnostic significance: AST

Answer 66

hepatocellular disorders (viral hepatitis, cirrhosis), skeletal muscle disorders (muscular dystrophies, inflammatory conditions), pulmonary embolism

Question 67

Source of error: AST

Answer 67

Hemolysis stable at room temp for 48 hrs 3–4 days at refrigerated temperatures

Question 68

Specimen Collection: AST

Answer 68

serum

Question 69

Assay: Karmen method AST

Answer 69

``` 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+. ```

Question 70

Reference range: AST

Answer 70

5–35 U/L (37°C)

Question 71

Alanine Aminotransferase (ALT)

Answer 71

Glutamic pyruvic transaminase (SGPT or GPT) More specific than AST for liver disorders found in Liver tissue

Question 72

De Ritis ratio (AST/ALT)

Answer 72

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

Question 73

Diagnostic significance ALT

Answer 73

hepatic disorders

Question 74

Reference range ALT

Answer 74

7 - 45 U/L (37°C)

Question 75

Specimen Collection ALT

Answer 75

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

Question 76

Assay: ALT

Answer 76

Wroblewski and LaDue Method pH = 7.3 – 7.8 340 nm

Question 77

Alkaline Phosphatase (ALP)

Answer 77

found in all tissues of the body esp. at or near cell membranes Highest concentration in hepatocytes

Question 78

Diagnostic significance: ALP

Answer 78

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)

Question 79

Reference range: ALP

Answer 79

42-128 U/L (30°C) | (M/F 20-50 y/o)

Question 80

Specimen Collection: ALP

Answer 80

``` 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+ ```

Question 81

Source of error: ALP

Answer 81

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

Question 82

Assay: Bowers & MaComb (ALP)

Answer 82

p-nitrophenyl-phosphate (colorless) is hydrolyzed to p-Nitrophenol (yellow) 405 nm

Question 83

γ-Glutamyltransferase (GGT)

Answer 83

Present in serum and all cells except muscle | kidney, brain, prostate, pancreas, liver

Question 84

Diagnostic significance: GGT

Answer 84

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

Question 85

Assay for enzyme activity: GGT

Answer 85

γ-glutamyl-p-nitroanilide is most widely accepted substrate used in GGT analysis Absorbance @ 405-420 nm

Question 86

Specimen collection:

Answer 86

Serum

Question 87

Source of error: GGT

Answer 87

stable for 1 week at 4°C | hemolysis not a concern

Question 88

Reference range: GGT

Answer 88

male: 6–55 U/L (37°C) female: 5–38 U/L (37°C)

Question 89

Amylase (AMS)

Answer 89

acinar cells of pancreas & salivary glands

Question 90

Diagnostic significance: AMS

Answer 90

Nonspecific finding acute pancreatitis disorders causing salivary gland lesions (mumps, parotitis) intraabdominal diseases

Question 91

Assay for enzyme activity: AMS

Answer 91

``` Requires Ca2+ and Cl- for activation Immunoassay Interference: opiates, morphine Serum / urine stable RT for 1 week or 4oC for 2 months ```

Question 92

AMY Methodologies

Answer 92

Maltotetraose reaction Used in many instruments Most common AMY → Maltose phosphorylase → β-Phosphoglucose mutase → Glucose-6-phosphate

Question 93

Reference Range: AMS

Answer 93

Serum: 40-140 U/L Urine: 24-400 U/L

Question 94

Lipase (LPS)

Answer 94

primarily in pancreas | also in stomach & small intestine

Question 95

Diagnostic significance: LPS

Answer 95

acute pancreatitis other intraabdominal diseases (penetrating duodenal ulcers, perforated peptic ulcers, intestinal obstruction, acute cholecystitis)

Question 96

Assay for enzyme activity: LPS

Answer 96

turbidimetric (simple and rapid) Colorimetric (perioxidase or glycerol kinase) Enzymatic LPS reactions have largely replaced titrimetric and turbidimetric methodologies

Question 97

Source of error: LPS

Answer 97

stable in serum for 1 week at room temperature & 3 weeks at 4°C; hemolysis

Question 98

Reference range: LPS

Answer 98

<45 U/L | LPS 🡪 Glycerol kinase 🡪 L-α-glycerophosphate kinase 🡪 Peroxidase

Question 99

Acid Phosphatase (ACP)

Answer 99

prostate, bone, liver, spleen, kidney, erythrocytes, platelets

Question 100

Diagnostic significance: ACP

Answer 100

prostatic carcinoma, hyperplasia of prostrate, prostatic surgery, osteoclasts, Paget’s disease, breast cancer with bone metastases, Gaucher’s disease

Question 101

Assay for enzyme activity: ACP

Answer 101

same techniques as in alkaline phosphatase, except performed in an acid pH

Question 102

Reference range: ACP

Answer 102

prostatic ACP: 0–3.5 ng/mL

Question 103

Source of error: ACP

Answer 103

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