Enzyme and Rate Analysis

Question 1

Define metabolism

Answer 1

An integrated series of enzymatic reactions

Question 2

Clinical enzymology is

Answer 2

The application of the science of enzymes to the diagnosis and treatment of disease

Question 3

Enzymes are…

Answer 3

Proteins with catalytic properties (ability to accelerate chemical reactions)

Question 4

6 classes of enzyme

Answer 4

1. oxidoreductase
2. transferase
3. hydrolase
4. lyase
5. somerase
6. ligase

Question 5

Denaturation is

Answer 5

The disruption of a protein’s structure, resulting in a loss of activity

Question 6

True or false: denaturation is reversible

Answer 6

Removal of the denaturing agent may allow reversal of the denaturing process if the extent of denaturation is minimal. If the process has been prolonged or severe, however, there may be irreversible loss of activity.

Question 7

Describe the effect of temperature on enzyme activity.

Answer 7

In general, increased temperatures denature enzymes. At room temperature, heat inactivation takes place at an appreciable rate. At 60 degrees C, heat inactivation occurs almost instantaneously for most enzymes, except polymerases. Polymerases retain activity up to 90 degrees C, a property that is taken advantage of with PCR. Low temperatures are used to preserve enzyme activity.

Question 8

List 3 factors that denature proteins/enzymes

Answer 8

1. Increased temperature
2. Extremes of pH
3. Chemical additions - urea, detergents

Question 9

How do chemicals such as urea and detergents inactivate enzymes?

Answer 9

By disruption of hydrogen bonds and hydrophobic interactions

Question 10

Enzymes are globular structures with ________ surfaces and _________ interiors.

Answer 10

Enzymes are globular structures with POLAR surfaces and HYDROPHOBIC interiors.

Question 11

What are the important properties of an enzyme’s active site?

Answer 11

4 properties
1. Active site generally small compared to the overall size of the enzyme (5% of the total amino acids or less)
2. Structure of the active site determines the specificity of the substrate that can bind
3. Active site is usually in a cleft or crevice of the protein
4. Attraction between active site and substrate is usually non-covalent ie hydrogen bonding van der Waals forces, hydrophobic or electrostatic interactions

Question 12

Define isoenzyme/isozyme. What is NOT included in this definition?

Answer 12

Multiple forms of enzymes arising from genetically determined differences in primary structure. Not applicable when students are due to post-translational or other non- genetic modifications

Question 13

Describe the genetic origins of enzyme variants (isoenzymes) and provide examples for each type.

Answer 13

1. Isoenzymes due to multiple gene loci for the same enzyme eg LD, CK, alpha-amylase and ALP - true isoenzymes
2. Isoenzymes due to different alleles for the same enzyme eg placental ALP or G6PD.
3. Hybrid isoenzymes - enzyme subunits are derived from different loci or different alleles, eg mixed MB dimer of CK, or LD-2, LD-3 and LD-4 of LD.

Question 14

The existence of multiple forms of enzymes has several important implications. List some.

Answer 14

1. Pharmacology - explains why individuals will have different sensitivities to drugs
2. Pathology - explains the existence of some hereditary metabolic diseases
3. Diagnostics - enables more sensitive and specific enzyme analysis of body fluids
4. Physiology - recognition of organ specific patterns of metabolism

Question 15

Define isoform

Answer 15

Post-translational modification of enzymes gives rise to isoforms. An example of non- genetic variation producing multiple forms of an enzyme

Question 16

Give examples of how post-translational modification of enzymes may result in different isoforms

Answer 16

1. De-amidation - the removal of amide residues (in vivo) from amylase or carbonic anhydrase
2. In vitro - glutathione normally exists in reduced form in intact cells. In haemolysed samples, glutathione becomes oxidised when it leaves the cell. Oxidised glutathione can then cause oxidation of vulnerable sulfhydryl groups in enzymes such as adenosine deaminase, acid phosphatase and phosphoglucomutase. Oxidation of sulfhydryl groups alters the enzyme’s charge.
3. Isoforms may be created as cells clear enzymes normally. For example, myocardial and skeletal muscle cells have CK-MM and CK-MB isoenzymes which are modified on release into the circulation by sequential removal of lysine by carboxypeptidase.
4. Enzymes may have non-protein components modified. For example, glycoproteins that have their carbohydrate moiety removed or otherwise altered may have different properties. An example is removal of terminal sialic acid groups from liver and bone ALP resulting in different electropheretic mobility of these isoforms.
5. Enzyme molecules can aggregate with each other to produce multiple forms with different molecular weights and activities eg cholinesterase; the heaviest component is the most active
6. Enzyme molecules can aggregate with non-enzyme proteins eg immunoglobulins to form macro-enzymes/complexes eg LD, CK, troponin, amylase

Question 17

Patterns of isoenzyme expression change during development. Give two examples

Answer 17

LD and CK expression changes through the early part of fetal development until the sixth month of intrauterine life when it resembles that of differentiated muscle
Liver aldolase has 3 isoenzymes - A, B and C - which are all detectable in fetal liver at different times, however at birth only the adult form - B - is detectable.

Question 18

Give examples in health and disease where adult tissues may express fetal distributions of isoenzymes

Answer 18

1. In regenerating tissues, where it may reflect relaxation of control systems in rapidly dividing cells
2. In progressive muscular dystrophies where it suggests failure to develop or mature normally
3. In malignancy, where it implies de-differentiation and therefore usually a worse prognosis

Question 19

Define an international unit

Answer 19

The quantity of enzyme that catalyses the reaction of 1micromol of substrate per minute. The unit of enzyme activity.

Question 20

Name some variables that affect the rate of a reaction

Answer 20

Temperature, pH, type of buffer, ionic strength, concentration of enzyme, concentration of substrate, nature of substrate, presence of cofactors, activators and inhibitors

Question 21

T or F. In a coupled enzyme reaction, the second reaction must be rate limiting.

Answer 21

FALSE. The first reaction (involving the enzyme activity we wish to measure) must be rate limiting.