ENZYMES GENERAL CLASSIFICATION OF ENZYMES:

Biologic proteins that catalyze biochemical reactions 1. Oxidoreductases - removal or addition of

without altering the equilibrium point of the reaction or electrons (reduction-oxidation ["redox"]
being consumed or changed in composition. reaction.)
Examples:
ENZYME NOMENCLATURE
(a) oxidase - cytochrome oxidase
✓ Practical or Trivial Name
(b) dehydrogenase
✓ Systematic Name
✓ lactate dehydrogenase (LDH)
A. PRACTICAL/TRIVIAL NAME
A1. According to the name of the substrate with the ✓ malate dehydrogenase (MDH)
addition of the suffix “ase”. ✓ isocitrate dehydrogenase (ICD)
2. Transferase - catalyze the transfer of a chemical
Examples: group from one substrate to another.
Examples:
✓ Enzymes acting on lipids – lipase
(a) aspartate aminotransferase (AST)
✓ Enzymes acting on proteins – protease
(b) alanine aminotransferase (ALT)
A2. According to the type of reaction they catalyzed. (c) creatine kinase (CN) / creatine phosphokinase
(CPK)
Examples:
(d) gamma glutamyl transferase (GGI)
✓ Transfer of amino group from substrate to (e) ornithine carbamoyl transferase (OCT)
another – transferase 3. Hydrolase - hydrolyze the splitting of a bond by
✓ Transfer to phosphate group from a high energy the addition of water (hydrolysis reaction)
phosphate compound to its substrate – kinase Examples:
✓ Effect of hydrolysis on phosphate esters – (a) Esterases
phosphatase ✓ acid phosphatase (ACP)
✓ Removal of hydrogen atoms from its substrate – ✓ alkaline phosphatase (ALP)
dehydrogenase ✓ cholinesterase (CLS)
B. SYSTEMIC NAME ✓ lipase (LPS)
B1. According to the numerical designation given by the (b) peptidases
Enzyme Commission (E.C.) ✓ trypsin (PTS)
✓ pepsin (PPS)
Examples:
✓ leucine aminopeptidase (LAP)
✓ E. C. 1. 1. 1. 7 for lactate dehydrogenase (c) glycosidase
✓ E. C. 3. 2. 1 .1 for amylase ✓ amylase, (kAMS)
✓ E. C. 2. 6.1. 2 for alanine aminotransferase ✓ amylo 1,6 glycosidase
✓ galactosidases
The first number defines the class to which the enzyme 4. Lyases - remove groups from substrate without
belongs, while the next two numbers indicate subclass hydrolysis, leaving only double bonds in the
and sub class to which the enzyme is assigned. The last molecular structure of the product.
number Is a specific serial number to each enzyme In its Examples:
sub-class. (a) aldolases
(b) glutamate decarboxylase
(c) pyruvate decarboxylase
(d) tryptophan decarboxylase
5. Isomerases - catalyzes the intramolecular
rearrangement of the substrate compound.
Examples:
(a) glucose phosphate isomerase
(b) ribose phosphate isomerase
6. Ligases (Synthetases) - joins two substrate ENZYME KENETICS:
molecules together using the energy released An enzyme (E) catalyses a reaction by combining with its
from hydrolyzing a pyrophosphate bond to a substrate (S) to create an enzyme—substrate complex
high-energy phosphate compound. (ES). The enzyme-substrate complex according to
Michaelis and Menten can either dissociate back to E + S
TERMS ASSOCIATED WITH ENZYMES: or breakdown to product (P) and free enzyme (provided
• Holoenzyme -an active substance formed by that the product has a low affinity for the enzyme).
combination of a coenzyme (cofactor) and
apoenzyme.
• Apoenzyme - the protein portion subject to
denaturation, in which the enzyme loses its THE MICHAELIS-MENTEN EQUATION GIVES THE MEANS
activity. Catalytically inactive protein when TO DETERMINE TOTAL ENZYME CONCENTRATION IN
cofactor is removed. They are heat labile and SERUM AND OTHER BODY FLUIDS
dialyzable.
• Isoenzyme - enzymes present in an individual Accurately describes virtually all single-substrate
with similar enzymatic activity but differ in their enzyme-catalyzed reactions and many bisubstrate
physical biochemical and immunologic reactions in which the concentration of one substrate is
characteristics. constant throughout the course of the reaction.
• Metalloenzyme - enzyme whose metal ions are MICHAELIS-MENTEN CONSTANT
intrinsically part the molecule such as catalases V= Vmax (S) / Km + (S)
and cytochrome oxidase.
Where:
• Proenzyme - inactive precursor of enzymes, also
referred to as zymogens. ✓ V = Velocity of the reaction
• Substrates - substances acted upon by the ✓ Vmax = Maximum velocity
enzymes which are specific for each of their ✓ S = Substrate concentration
particular enzyme. ✓ Km = Michaelis-Menten constant
• Cofactors - these are non-protein TYPES OF SPECIFICITY
substance/compounds needed by an enzyme • Absolute Specificity – enzymes combine with
before enzymatic activity can be manifested. only one substrate and catalyzes only one
Cofactors are thermostable and dialyzable. corresponding reaction
COFACTOR • Group Specificity – enzymes combining with all
1. Organic molecule - Coenzyme. substrates containing a particular chemical
• It hastens enzymatic reaction but undergoes group
a change or is consumed to another product. • Bond Specificity – enzymes are specific to
Examples: chemical bonds
✓ NAD – nicotinamide Adenine • Stereoisomeric Specificity – enzymes that
dinucleotide predominantly combine with only one optical
✓ NADP - nicotinamide Adenine isomer of a certain compound
dinucleotide phosphate
2. Metal ion – Activator
In such, the metal ion may serve as:
• a bridge to hold the substrate and enzyme
together
• the primary catalytic center
• stabilizing agent in the conformation for
catalytic activity.
Examples:
✓ Amylase - Cl, Br / LDH - Zn2 /
Lipase - Ca++
ENZYME SPECIFICITY TYPES OF REACTION ORDER:
EMIL FISHER'S LOCK AND KEY THEORY 1. Zero Order Reaction - is the rate of reaction
linear with time, independent of concentration
It is based on the rigid enzyme molecule into which the
of substrate and directly proportional to enzyme
substrate fits. The shape of the key (substrate) must
concentration.
conform into the lock (enzyme).
2. First Order Reaction - the rate of reaction is
determined by the concentration of substrate.as
well as of enzymes (the rate of reaction changes
continuously with time as the substrate is
consumed.
FACTORS AFFECTING ENZYME REACTIONS:
• Enzyme concentration. - An increase in the
concentration of enzyme produces an increase in
the rate of reaction, provided that the other
conditions remain the same and that a constant
but excess amount of substrate Is present.
Meaning, if the amount of enzyme is doubled,
the reaction proceeds twice as fast.
• Substrate Concentration - An increase in the
concentration of substrate produces also an
increase in the rate of reaction, provided all
KOSHLAND'S INDUCED FIT THEORY other conditions are kept constant However, the
rate of the reaction reaches a maximal value at a
It is based on the attachment of a substrate to the active
particular concentration of substrate, and higher
site of an enzyme, which then causes conformational
concentrations of substrate do not result in
changes in the enzyme. This theory Is more acceptable
increased rate of reaction (Saturation kinetics).
because the protein molecule Is flexible enough to allow
• Temperature - The rate of any chemical reaction
conformational changes and also allow some
is usually increased 2-3 times for every I0
explanation on the influence of hormones on enzymatic
degrees Celsius rise in temperature.
activity.
• Hydrogen Ion Concentration or pH - Enzymatic
reactions proceed at their fastest rate at an
optimum pH and are considerably slowed or
even stopped at higher or lower pH values.
ENZYME INHIBITION: PITFALLS IN ENZYME ASSAYS
• Competitive Inhibitor - These are substances • Hemolysis cause falsely elevated values due to
that compete with the substrate for enzyme the release of enzymes from red blood cells.
binding because they are chemically analogous • Serum rather than plasma is the preferred
to the substrate and bind to the active sites of specimen due to the adverse effects of
enzymes. anticoagulants on enzyme activity.
• Non- competitive Inhibitor. - These are • Lactescent or milky serum causes variable
substances that do not resemble the substrate absorption by the spectrophotometer.
and bind to the enzyme in areas other than the • Storage
active site. STORAGE OF SAMPLES
• Uncompetitive Inhibition – inhibits enzyme by • Most enzymes are stable at 6C for at least 24 hrs
binding to the enzyme substrate complex. • Few enzymes are inactivated at refrigerator
ENZYME INDUCTION: temp (LD 4 and 5)
This phenomenon states that a certain enzyme has the QUALITY CONTROL PROGRAM FOR ENZYME ASSAYS
ability to adapt to their biochemical systems. • Strict adherence to zero-order kinetics
TYPES OF ENZYME ASSAYS • Proportionality with increments of sample
1. Endpoint Analysis • Use of pooled frozen serum or stable reference
• Reaction is initiated by addition of substrate materials as controls
• Reaction is allowed to proceed for a period • Replicate measurements to evaluate precision of
of time assays
• Measurement is done at the end of the
reaction
• Disadvantage: underestimation of the “true”
enzyme activity and linearity of reaction
cannot be observed
2. Multi-point and Kinetic Assay
• Change in concentration of the indicator
substance at several intervals
• Continuous measurement of change in
concentration as function of time
3. Use of Coupled Reactions:
1. Enzymatic activity is measured by coupling
the activity with colorimetric reaction
UNITS FOR EXPRESSING ENZYME ACTIVITY
1. International Unit (I.U. or U)
- Equivalent to the amount of enzyme that
catalyzes the conversion of 1 micromole of
substrate per minute under controlled
conditions.
2. Katal Unit (K.U.)
- Equivalent to the amount of enzyme that
catalyzes the conversion of 1 mole of
substrate per second under controlled
conditions.
MEANS OF MEASURING ENZYME ACTIVITY
1. Change in Coenzyme Concentration
2. Increase in Product Concentration
3. Decrease in Substrate Concentration