Name: Michelle Mae A.

Juliana
Course and Year: BMLS 3
Code: 5159
Instructor: Giles D. Dumangeng, RMT
Date: March 29, 2022

Assignment – Enzymes (98)

I. Description (42)
1. What is an enzyme? (2)
 An enzyme is a specific biologic protein that catalyze biochemical reactions
without altering the equilibrium point of the reaction or being consumed or
changed in composition. Each enzyme catalyzes a single reaction or a limited
number of chemical reactions, and it is specific for a substrate that it converts to a
defined product.

2. What is an active site and allosteric site? (4)

 An active site is a 3-dimensional protein structure, a water-free cavity, where the
substance on which the enzyme acts (the substrate) and interacts with particular
charged amino acid residues. Whereas, an allosteric site is a cavity other than the
active site that may bind regulator molecules and, thereby, be significant to the
basic enzyme structure.

3. What is a cofactor? Also, give the types of cofactors and their definition. (6)
 Cofactor is a nonprotein molecule that may be necessary for enzyme activity.
 Types of cofactors:
1) Coenzymes – organic compound (second substrates); increasing its
concentration will increase the velocity of an enzymatic reaction; it is
essential to achieve absolute enzymatic activity; examples include NAD and
NADP
2) Activators – inorganic ions which alters the spatial configuration of the
enzyme for proper substrate binding; examples include calcium, zinc,
chloride, magnesium, and potassium
3) Metalloenzymes – inorganic ion attached to a molecule; examples include
catalase and cytochrome oxidase

4. What are the major classifications of clinically significant enzymes? Give their
definitions and give two examples for each. (12)
1) Oxidoreductases
 Catalyze an oxidation–reduction reaction between two substrates
  Examples: Lactate dehydrogenase, Glutamate dehydrogenase
2) Transferases
 Catalyze the transfer of a group other than hydrogen from one substrate to
another
 Examples: Aspartate aminotransferase, Alanine aminotransferase
3) Hydrolases
 Catalyze hydrolysis of various bonds
 Examples: Alkaline phosphatase, Acid phosphatase
4) Lyases
 Catalyze removal of groups from substrates without hydrolysis; the product
contains double bonds
 Examples: Aldolase, Glutamate decarboxylase
5) Isomerases
 Catalyze the interconversion of geometric, optical, or positional isomers
 Examples: Triosephosphate isomerase, Glucose phosphate isomerase
6) Ligases
 Catalyze the joining of two substrate molecules, coupled with breaking of the
pyrophosphate bond in adenosine triphosphate (ATP) or a similar compound
 Examples: Glutathione synthetase, Synthase

5. What factors affect enzyme activity? Give five and a simple explanation for each.
(10)
1) Enzyme concentration
 The higher the enzyme concentration, the faster is the reaction, because more
enzyme is present to bind with the substrate.
2) Substrate concentration
 With the amount of enzyme exceeding the amount of substrate, the reaction
rate steadily increases as more substrate is added
 However, when the substrate concentration reaches a maximal value, higher
concentration of substrate no longer results in increased rate of reaction
(saturation kinetics).
3) Hydrogen Ion Concentration or pH
 Most physiologic reactions occur in the pH range of 7 to 8
 Extreme pH level may denature an enzyme of influence its ionic state
resulting in structural change or change in the charge of amino acid residue in
the active site.
4) Storage
 Low temperatures (refrigeration/freezing) render enzymes reversibly inactive
  Repeated freezing and thawing tend to denature proteins and should be
avoided
 -20°C is the ideal temperature for preservation of enzymes (longer period of
time)
 2°C - 8°C is the ideal storage temperature for substances and coenzymes
 22°C or room temperature is the ideal storage of LDH (LD4 and LD5)
5) Temperature
 Enzymes are active at 25°C, 30°C, or 37°C
 37°C is the optimum temperature for enzymatic activity
 Increasing temperature usually increases the rate of a chemical reaction by
increasing the movement of molecules
 The rate of denaturation increases as the temperature increases, and is usually
significant at 40°C to 50°C
 60-65°C may result to inactivation of enzymes
 Temperature Coefficient (Q10) means for every 10°C increase in temperature,
there will be a two-fold increase in enzyme

6. In what ways can enzymes be measured? (4)

 Enzymes are measured in terms of change in the substrate concentration, change
in the product concentration, and change in coenzyme concentration.
 To measure the extent of enzymatic reactions, two general methods may be used:
1) Fixed-Time
 The reactants are combined, the reaction proceeds for a designated time,
the reaction is stopped (usually by inactivating the enzyme with a weak
acid), and a measurement of the amount of reaction that has occurred is
made.
 The reaction is assumed to be linear over the reaction time; the larger the
reaction, the more the enzyme present.
2) Continuous Monitoring/Kinetic Assay
 Multiple measurements of changed in absorbance are made during the
reaction, either at specific time intervals (usually every 30 or 60 seconds)
or continuously by a continuous-recording spectrophotometer.
 It is preferred than fixed-time method because the linearity of the reaction
may be more adequately verified.

7. What are the causes of elevated serum enzyme levels? (4)

 Impaired removal of enzyme from plasma
 Increased permeability of cell membrane
 Increased in the number of cells or the production of cells
 Increased in the normal cell turnover
  Decreased clearance of enzymes from the circulation
 Tissue necrosis and degeneration – death of enzyme-containing cells

II. Essay. (26)

1. Explain the process of naming enzymes with the IUB system. (5)
 The IUB system assigns a systematic name to each enzyme, defining the substrate
acted on, the reaction catalyzed, and, possibly, the name of any coenzyme
involved in the reaction. In addition to naming enzymes, the IUB system
identifies each enzyme by an EC numerical code containing four digits separated
by decimal points. The first digit places the enzyme in one of the six classes,
including Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, and
Ligases. The second and third digits of the EC code number represent the subclass
and subsubclass of the enzyme, respectively, divisions that are made according to
criteria specific to the enzymes in the class. Lastly, the final number is the serial
number specific to each enzyme in a subsubclass.

2. How do enzymes speed up the rate of chemical reactions? (5)

 An enzyme accelerates the rate of chemical reactions by reducing the activation
energy level that the substrate must reach for the reaction to occur. An enzyme
combines only with one substrate and catalyzes only one reaction. A chemical
reaction may occur spontaneously if the free energy is higher for the substrate
than the product.

3. Explain the first-order kinetics. (5)

 The substrate readily binds to free enzymes at a low substrate concentration. With
the amount of enzyme exceeding the amount of substrate, the reaction rate
steadily increases as more substrate is added because the reaction rate is directly
proportional to substrate concentration.

4. Explain the zero-order kinetics. (5)

 The reaction rate depends only on enzyme concentration because the substrate
concentration is high enough to saturate all available enzymes, and the reaction
velocity reaches its maximum. When the product is formed, the resultant free
enzyme immediately combines with an excess free substrate.

5. Explain the process of competitive inhibition. (2)

 Competitive inhibitors physically bind to the active site of an enzyme and
compete with the substrate for the active site. With a substrate concentration
significantly higher than the concentration of the inhibitor, the inhibition is
reversible because the substrate is more likely than the inhibitor to bind the active
 site and the enzyme has not been destroyed. In addition, dilution of serum results
to reduction in the concentration of this inhibitor, thus increasing the rate of
reaction.

6. Explain the process of noncompetitive inhibition. (2)

 A noncompetitive inhibitor binds an enzyme at a place other than the active site
and may be reversible in that some naturally present metabolic substances
combine reversibly with certain enzymes. Noncompetitive inhibition also may be
irreversible if the inhibitor destroys part of the enzyme involved in catalytic
activity. Because the inhibitor binds the enzyme independently from the substrate,
increasing substrate concentration does not reverse the inhibition.

7. Explain the process of uncompetitive inhibition. (2)

 Uncompetitive inhibition is another kind of inhibition in which the inhibitor binds
to the ES complex – increasing substrate concentration results in more ES
complexes to which the inhibitor binds and, thereby, increases the inhibition. The
enzyme-substrate–inhibitor complex does not yield a product

III. Summary (30)

1. Creatine kinase
Function:
 Catalyzes the transfer of a phosphate group between creatine phosphate and
adenosine diphosphate
 Involved in the storage of high-energy creatine phosphate in the muscles
Major tissue source/s:
 Brain tissue, skeletal muscles, and heart muscles
Isoenzymes:
 CK-BB (brain type)
 CK-MB (hybrid-type)
 CK-MM (muscle type)
Causes of elevation/s:
 CK-MM
o Myocardial infarction
o Skeletal muscle disorder
o Muscular dystrophy
o Polymyositis
o Hypothyroidism
o Malignant hyperthermia
o Physical activity
o Intramuscular injection
  CK-MB
o Myocardial infarction
o Myocardial injury
o Ischemia
o Angina
o Inflammatory heart disease
o Cardiac surgery
o Duchenne-type muscular dystrophy
o Polymyositis
o Malignant hyperthermia
o Reye’s syndrome
o Rocky Mountain spotted fever
o Carbon monoxide poisoning
 CK-BB
o Central nervous system shock
o Anoxic encephalopathy
o Cerebrovascular accident
o Seizure
o Placental or uterine trauma
o Carcinoma
o Reye’s syndrome
o Carbon monoxide poisoning
o Malignant hyperthermia
o Acute and chronic renal failure
Method/s of measurement:
 Tanzer-Gilbarg Assay (forward-direct method)
 Oliver-Rosalki (reverse/indirect method)

2. Lactate dehydrogenase
Function:
 Catalyzes the interconversion of lactic and pyruvic acids
 A hydrogen-transfer enzyme that uses the coenzyme nicotinamide
dinucleotide (NAD+)
Major tissue source/s:
 Heart, liver, skeletal muscle, kidney, and erythrocytes
Isoenzymes:
 LD-1 (HHHH)
 LD-2 (HHHM)
 LD-3 (HHMM)
  LD-4 (HMMM)
 LD-5 (MMMM)
 LD-6 (Alcohol dehydrogenase)
Causes of elevation/s:
 LD-1 (HHHH)
o Myocardial infarction
o Hemolytic anemia
 LD-2 (HHHM)
o Megaloblastic anemia
o Acute renal infarct
o Hemolyzed specimen
 LD-3 (HHMM)
o Pulmonary embolism
o Extensive pulmonary pneumonia
o Lymphocytosis
o Acute pancreatitis
o Carcinoma
 LD-4 (HMMM)
o Hepatic injury or inflammation
 LD-5 (MMMM)
o Skeletal muscle injury
Method/s of measurement:
 Wacker method (forward/direct reaction)
 Wrobleuski La Due (reverse/indirect reaction)
 Wrobleuski Cabaud
 Berger Broida

3. Aspartate aminotransferase
Function:
 It is involved in the transfer of amino group between aspartate and α-keto
acids with the formation of oxaloacetate and glutamate.
Major tissue source/s:
 Cardiac tissue, liver, and skeletal muscle
 Other sources: kidney, pancreas, erythrocytes
Isoenzymes:
 Cytoplasmic AST – predominant form in serum
 Mitochondrial AST – increased in cellular necrosis
Causes of elevation/s:
 Acute myocardial infarction
 Pulmonary embolism
  Congestive heart failure
 Viral hepatitis
 Cirrhosis
 Skeletal muscle disorder
 Muscular dystrophy
 Inflammatory conditions
Method/s of measurement:
 Karmen method – It uses malate dehydrogenase (MD) and monitors the
change in absorbance at 340 nm.

4. Alanine aminotransferase
Function:
 It catalyzes the transfer of an amino group from alanine to α-ketoglutarate
with the formation of glutamate and pyruvate
Major tissue source/s:
 Liver
 Other sources: kidney, pancreas, red blood cells, heart, skeletal muscles, lungs
Isoenzymes:
Causes of elevation/s:
 Toxic hepatitis
 Wolff-Parkinson White syndrome
 Chronic alcoholism
 Hepatic cancer
 Reye’s syndrome
 Viral hepatitis
Method/s of measurement:
 Coupled enzymatic reaction

5. Alkaline phosphatase
Function:
 Belongs to a group of enzymes that catalyze the hydrolysis of various
phosphomonoesters at an alkaline pH.
 Nonspecific enzyme capable of reacting with many different substrates.
 Liberate inorganic phosphate from an organic phosphate ester with the
concomitant production of an alcohol.
Major tissue source/s:
 Intestine, liver, bone, spleen, placenta, and kidney
Isoenzymes:
 Liver ALP
 Bone ALP
 Intestinal ALP
  Placental ALP
Causes of elevation/s:
 Biliary tract obstruction
 Hepatocellular disorders – hepatitis, cirrhosis
 Paget’s disease (osteitis deformans) – highest elevation
 Other bones disorders: osteomalacia, rickets, hyperparathyroidism, osteogenic
sarcoma
 Healing bone fractures, bone growth
 Pregnancy
Method/s of measurement:
 Bowers and McComb (Szasz modification)
 Bodansky
 Shinowara
 Jones
 Reinhart
 King and Armstrong
 Bessy, Lowry & Brock
 Huggins and Talalay
 Moss
 Klein, Babson & Read

6. Acid phosphatase
Function:
 Catalyzes the same type of reactions. The major difference between ACP and
ALP is the pH of the reaction.
 ACP functions at an optimal pH of approximately 5.0.
Major tissue source/s:
 Prostate
 Other sources: Bone, liver, spleen, kidney, erythrocytes, and platelets
Isoenzymes:
 Prostatic ACP - Major fraction in serum
 Erythrocyte ACP
 Tartrate-resistant ACP (TRAP)
Causes of elevation/s:
 Prostatic cancer
 Paget’s disease
 Breast cancer with bone metastases
 Gaucher’s disease
 Urinary tract obstruction
 Acute urinary retention
 Extensive prostatic massage
  Prostatic inflammation
 Infarction/ischemia
 Prostatic manipulations (needle biopsy and cystoscopy)
 Idiopathic thrombocytopenic purpura
Method/s of measurement:
 Gutman and Gutman
 Shinowara
 Babson, Read & Phillips
 Roy and Hillman

7. Gamma-glutamyltransferase
Function:
 Involved in the transfer of the γ-glutamyl residue from γ-glutamyl peptides to
amino acids, H2O, and other small peptides.
 Glutathione serves as the γ-glutamyl donor.
Major tissue source/s:
 Kidney, brain, prostate, pancreas, and liver
Isoenzymes:
Causes of elevation/s:
 Biliary tract obstruction
Method/s of measurement:
 Continuous-monitoring or Fixed-point method

8. Amylase
Function:
 Catalyze the breakdown of starch and glycogen.
 Requires calcium and chloride ions for its activation.
Major tissue source/s:
 Acinar cells of the pancreas, and the salivary glands
 Other sources: Skeletal muscle, small intestine, fallopian tubes
Isoenzymes:
 P-type iso-amylase - pancreatic amylase
 S-type iso-amylase - salivary amylase
Causes of elevation/s:
 Acute pancreatitis
 Salivary gland lesions – mumps and parotitis
 Intra-abdominal diseases – perforated peptic ulcer, intestinal obstruction,
cholecystitis
 Ruptured ectopic pregnancy
 Mesenteric infarction
 Acute appendicitis
  Renal insufficiency
 Diabetic ketoacidosis
 Hyperamylasemia
 Macroamylasemia
Method/s of measurement:
 Saccharogenic
 Amyloclastic
 Chromogenic
 Continuous monitoring/Coupled enzyme

9. Lipase
Function:
 Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids.
 Catalyzes the partial hydrolysis of dietary triglycerides in the intestine to the
2- monoglyceride intermediate, with the production of long-chain fatty acids.
Major tissue source/s:
 Pancreas
 Other sources: stomach, and small intestine
Isoenzymes:
 L1
 L2 – most clinically specific and sensitive
 L3
Causes of elevation/s:
 Acute pancreatitis
 Penetrating duodenal ulcers
 Perforated peptic ulcers
 Intestinal obstruction
 Acute cholecystitis
Method/s of measurement:
 Cherry-Crandall method (reference method)
 Tietz and Fiereck
 Peroxidase coupling

10. Glucose-6-phosphate dehydrogenase

Function:
 Catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate or the
corresponding lactone.
Major tissue source/s:
 Adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, and
erythrocytes.
Isoenzymes:
Causes of elevation/s:
 G-6-PD deficiency
 Hemolytic anemia
 Megaloblastic anemias
Method/s of measurement:
 Enzymatic method