Commonly used and recommended name of an enzyme molecule

The name of enzyme can readily tell the specific function as it is included in the name and can also tell the specific substrate of a particular enzyme.

Enzymes acting on lipids

Enzymes acting on proteins

transfer of amino group from substrate to another

transfer to phosphate group from a high energy phosphate compound to its substrate

effect of hydrolysis on phosphate esters

removal of hydrogen atoms from its substrate

Standardized by the Enzyme Commission specifically by International Union of Biochemistry

E. C. 1. 1. 1. 7

E. C. 3. 2. 1 .1

E. C. 2. 6.1. 2

The first number defines the _____ to which the enzyme belongs

the next two numbers indicate _____, to which the enzyme is assigned

last number is a _______ to each enzyme in its sub-class.

1. Oxidoreductases

"ODR"

2. Transferases

"COAAG"

oxidation reaction

involved in reduction reaction

oxidation-reduction reaction

3. Hydrolases

"PEG"

4. Lyases

"GAPT"

5. Isomerases

"GR"

6. Ligases/Synthetases

removal or addition of electrons (reduction-oxidation ["redox"] reaction.)

catalyze the transfer of a chemical group from one substrate to another

hydrolyze the splitting of a bond by the addition of water (hydrolysis reaction)

remove groups from substrate without hydrolysis, leaving only double bonds in the molecular structure of the product.

catalyzes the intramolecular rearrangement of the substrate compound.

joins two substrate molecules together using the energy released from hydrolyzing a pyrophosphate bond to a high-energy phosphate compound.

** Counterpart of lyases

An enzyme molecule could either be released as an active/inactive enzyme molecule

- an active substance formed by combination of a

coenzyme (cofactor) + apoenzyme.

- Readily functional and capable of catalyzing certain reaction

- the protein portion subject to denaturation, in which the enzyme loses its activity.

- Catalytically inactive protein when cofactor is removed.

- Heat labile and dialyzable.

- these are non-protein substance/compounds needed by an enzyme before enzymatic activity can be manifested

- Thermostable (heat stable) and dialyzable

Cofactor with organic molecule

Ex: NAD, NADP

Ex: Fe, Mg, Mn, Br, Cl molecule

Coenzyme + Apoenzyme

Cofactor with inorganic molecule

Sometimes activators could be made up of metal ion like metal irons

enzyme whose metal ions are intrinsically part the molecule such as catalases and cytochrome oxidase.

inactive precursor of enzymes, also referred to as zymogens

Initially released as inactive enzyme like pepsinogen in stomach which would require the presence of HCl so it can be converted to pepsin (active molecule)

enzymes present it an individual with similar enzymatic activity but differ in their physical biochemical and immunologic characteristics

Ex: Alkaline phosphatase – bone, placental, and liver

*** They are classified as ALP because they catalyze same type of reaction but under ALP, it has specific isoenzymes which differ in particular organ of origin, and different reactions when subjected to heat and exposed to chemicals.

Variety of enzyme molecule which has already undergone post-translational mutations in its genetic makeup specifically its sequence

substances acted upon by the enzymes which are specific for each of their particular enzyme.

site where substrate interconnects with enzyme

** enzyme + substrate

the rest of the site; area where no substrate is bound to enzyme molecule (where inhibitor or other compounds bind with enzyme molecule)

** inhibitor + enzyme

Cofactors

Organic molecule

It hastens enzymatic reaction but undergoes a change or is consumed to another product.

Metal ion

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.

- the ES complex would catalyze the reaction until the reactants are converted into products.

- An enzyme is neither consumed/changed so at the end of reaction, the enzyme will be liberated as free enzyme until all substrate molecules has already been consumed and converted.

- the ES complex would dissociate into separate substrate and enzyme molecule

Less specific:

- enzymes combine with only one substrate and catalyzes only one corresponding reaction

- will not look for certain bond/group

enzymes combining with all substrates containing a particular chemical group

will look for a compound containing an organic phosphate group

Ex: Phosphatases such as ALP and ACP (3. Hydrolases)

enzymes are specific to chemical bonds

Ex: Esterases – such as those involved in reesterification of lipids which looks for an ester bond (3. Hydrolases)

enzymes that predominantly combine with only one optical isomer of a certain compound

pertains to the capability of enzyme to determine a substrate according to its isomers

Ex: L –glucose and D – glucose – capable of identifying which type of stereoisomer of glucose there is

It is based on the rigid enzyme molecule into which the substrate fits.

The shape of the KEY (substrate) must conform into the LOCK (enzyme).

It is based on the attachment of a substrate to the active site of an enzyme, which then causes conformational changes in the enzyme.

This theory is more acceptable because the protein molecule Is flexible enough to allow conformational changes and also allow some explanation on the influence of hormones on enzymatic activity.

Factors affecting enzyme reactions

An increase in the concentration of enzyme produces an increase in the rate of reaction, provided that the other conditions remain the same (ex: no changes in temp/pH) and that a constant but excess amount of substrate Is present (ES complex would only happen in the presence of substrate). Meaning, if the amount of enzyme is doubled, the reaction proceeds twice as fast.

An increase in the concentration of substrate produces also an increase in the rate of reaction, provided all other conditions are kept constant. However, the rate of the reaction reaches a maximal value at a particular concentration of substrate, and higher concentrations of substrate do not result in increased rate of reaction (Saturation kinetics).

Q-10 - The rate of any chemical reaction is usually increased 2-3 times for every I0 degrees Celcius rise in temperature.

If the temp would be 47*C, this would result to

if the temp is about 27*C

Majority of enzymes are maintained up to ________ (above this will undergo denaturation).

the activity will stop

Enzymatic reactions proceed at their fastest rate at an optimum pH and are considerably slowed or even stopped at higher or lower pH values.

But, several enzymes will require different pH levels:

*** Acid phosphatase (ACP) – ___ pH (acidic)

But, several enzymes will require different pH levels:

*** Alkaline phosphatase (ALP) – ___ pH (basic)

is the rate of reaction linear with time, independent of concentration of substrate and directly proportional to enzyme concentration.

If in case all substrate has been bound to an enzyme molecule, and is independent of the substrate concentration. It is only dependent in enzyme concentration.

In enzyme determination activity, we follow ________ to know the actual activity of enzyme in the body based on the standardized concentration of substrate. The components of reaction must not be altered unnecessarily.

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.)

There are 2 factors involved: substrate and enzyme. Every time we increase the substrate and enzyme concentration, the rate of reaction will also increase. But, there will be a point where a substrate will reach its optimal/maximal act (saturation kinetics)

3 Factors to Determine the Type of Inhibition

These are substances that compete with the substrate for enzyme binding because they are chemically analogous to the substrate and bind to the active sites of enzymes

Competition between substrate and inhibitor. The enzyme will have an active site that would be the target of both substrate and inhibitor.

** reversible inhibition

substances that do not resemble the substrate and bind to the enzyme in areas other than the active site

The inhibitor doesn’t need to be chemically analogous to the substrate and it doesn’t target the active site.

Both reversible and irreversible – there’s still a competition between the substrate and inhibitor on which to bind first with the enzyme. If inhibitor is 1st to bind, this would cause disruption in the normal act. of enzymatic reaction.

inhibits enzyme by binding to the enzyme substrate complex

This inhibition is very effective in a way that even if there’s already an ES complex, the reaction would not yield product due to the presence of inhibitor.

The concentration of an enzyme is NOT directly measured. Rather, it is measured according to its activity.

* Reaction is initiated by addition of substrate

* Reaction is allowed to proceed for a period of time

* Measurement is done at the end of the reaction

Only single measurement is done

Disadvantage: underestimation of the “true” enzyme activity and linearity of reaction cannot be observed

Change in concentration of the indicator substance at several intervals

Continuous measurement of change in concentration as function of time (multiple readings are done at a certain period of time)

Example:

 ALP testing – test will be done for 2 mins and the reading will be done every 30 secs.

 Another is to get reading at certain time interval (3-5 absorbance reading within a particular reaction time)

Advantage 1: Discrepancy can be identified and parallel run can be done if the process is slightly questionable.

Advantage 2: No underestimation because the activity is derived based on the changes of absorbance.

Enzymatic activity is measured by coupling the activity with colorimetric reaction

Only additional; can be applicable on both end-point and continuous monitoring

- Significant in optical method such as the use of spectrophotometric reading of analytes to determine enzyme activity.

- The colorimetric reaction would help to identify if there are changes in the substrate, product, and cofactor

conventional unit

SI unit

Equivalent to the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under controlled conditions.

**** IU/L

Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second under controlled conditions.

*** mol/s

one of the most common method done in the laboratory

ex: Reduction of NAD → NADH measured ad 340nm

 Many enzymes bind to coenzyme (NAD) in which the reaction such as oxidation-reduction rxn would undergo spectrophotometric/optical changes specially if coupled with colorimetric rxn

In a reaction, reactants are converted into products so it will increase. It may be an end-point or multi-point analysis but the following conditions must always be met.

* Avoid any icteric, lipemic, or hemolyzed sample.

* There must be no chemical interferences

* The substrate must not be fully exhausted immediately during the incubation period. There must be enough amount of substrate to finish the reaction especially in end-point analysis.

Substrate will decrease because they are converted to products

Pitfalls in enzyme activity

Few enzymes are inactivated at refrigerator temp

QUALITY CONTROL PROGRAM FOR ENZYME ACTIVITY

Enzyme measurement is used for several purposes:

 To detect possible injury to a tissue where the enzymes are coming from

 To identify possibility of presence of abnormalities or absence of enzymes which may lead to a certain condition like inborn errors

Alkaline phosphatase – optimum activity seen in alkaline pH:

Acid phosphatase - optimum activity seen in acid pH:

only in pregnant individuals

** peak level: 16th-20th week of gestation

good marker for being pregnant but is not typically used and not specific.

Blood types A and AB

blood types ___ have LOWER placental isoenzyme

- released by the GIT cells

- individuals have significant differences according to blood type

Type B and O indiv – have higher. They have secretor genes so it is expected that their ALP is higher most importantly about after 2-3hrs of eating.

have HIGHER.

They have secretor genes so it is expected that their ALP is higher most importantly about after 2-3hrs of eating.

Released by liver cells specifically those cells lining the canaliculi significant in post-hepatic and obstructive liver disease

- Released by osteocytes

- Vary per age group

In bone isoenzymes, ______ (in period of rapid growth) – higher ALP because their osteocytes are actively replicating

In bone isoenzymes, ______ (>50yrs old) – have higher ALP due to increased possible damage to the osteocytes thereby releasing ALP in bloodstream

(most heat-labile) – readily be denatured upon exposure to heat

(most heat stable) – will remain in the sample even after heat exposure. It will still undergo denaturation at a higher temp. of >60-65oC.

inhibits the placental and intestinal ALP

inhibits bone ALP

inhibits liver and bone ALP

- seen in lung, breast, ovarian and gynecological cancers

- bone ALP co-migrator: same migration pattern of bone ALP in electrophoresis

- most heat STABLE, inhibited by phenylalanine reagent

- (variant of Regan)

- adenocarcinoma of the pancreas and bile duct, pleural cancer

- inhibited by L-leucine and phenylalanine

hepatoma/hepatocellular Carcinoma

For liver tissue sources, there are 2 types of ALP