Enzymes are protein catalysts responsible for most of the chemical reactions of the body

They are found in: cells and all tissues; serum to which gain access from injured cells, cells undergone stress

in disease states, caused in increased membrane permeability

caused by increase rates of intracellular synthesis

subsequent diffusion of enzymes

Enzymes found in serum will help physicians diagnose certain disease and aid in the monitoring of the disease condition

Increase serum levelsincreased release of enzyme from

sourceo necrosiso increased membrane permeability

increased size of tissue source of enzyme

impaired excretion of enzymeincreased enzyme synthesis

Decreased serum levelsdecreased formation

- Genetic - Acquired- Enzyme inhibition

poisoninglack of cofactors

Alkaline phosphatase – diagnose osseous and hepatobiliary diseases

Acid phosphatase – in the diagnosis of prostate cancers

Amylase and lipase – diagnosis of pancreatic disease

SGPT – liver diseases (hepatitis)

SGOT – cardiac and liver diseases

Catalyst A substance that enhances the rate of a

chemical reaction but is not permanently altered by the reaction

Decreases the activation of energy required for a chemical reaction and provides an alternative reaction pathway that requires less energy

Enzymes are neither produced or consumed in the reaction

Enzymes do not cause the reaction but enhances the reaction to occur

Enzymes are long sequences of amino acid

Enzymes are highly specific and produce only the expected product from given reactant or substrate

Enzymes acts on moderate pH and temperature

Enzyme possess the following:Active site – where the substrate fits

before converted to corresponding product

Allosteric site – which binds effector molecules or modifiers which regulates enzyme activity

Apoenzyme – are protein portion of the enzyme

Coenzyme – non-protein component; main participant during reaction with a substrate

help in enzymatic activity by orienting properly the substrate with the active site.

Coenzyme – organic cofactor: NADH, NADPH, FAD and FMN

Activator - usually metallic ions tightly bound to enzymes: Mg, Zn++, Cu++

Other enzymes are pro-enzymes or zymogens that are inactive but once released to their target sites they become activated.

Isoenzymes – enzymes with similar enzymatic activities but differ in their chemical structure and origins

They are unchanged during the course and termination of chemical reaction.

They demonstrate great specificity:Absolute – pyruvate kinaseGroup - phosphatasesBond - hydrolasesStereospecificity - transaminases

Enzyme binds to a single type of substrate because of complementary structures of the active site and substrate

Substrate’s overall shape and charge allows to interact with enzymes active site

Flexibility structure of protein is taken into account

Substrate does not precisely fit into the rigid active site instead a non-covalent interaction of enzyme-substrate change it thus conforming to the shape of active site to the shape of the substrate

Substrate ConcentrationMichaelis-Menten hypothesis: the rate of

substrate conversion to product is determined by substrate concentration and rate of dissociation of enzyme substrate complex

first-order kineticsAt constant enzyme concentration, the velocity or speed

of the enzyme reaction initially increases as the substrate concentration increases.

Reaction rate is dependent on the substrate concentration

Zero-order kineticsThe point at which the further addition of substrate does not anymore change the velocity of enzyme reaction.

The reaction is now dependent on the enzyme concentration

Km Represents the substrate concentration where the velocity is ½ the maximumRepresents the substrate concentration at which the enzyme yields half the possible maximum velocityIt is also the measure of affinity of the enzymes with its substrate

Enzyme concentrationThe higher the enzyme concentration the

higher is the reaction rate. (true only in zero-order kinetics)

pHoptimal pH varies with each enzyme

Temperaturemost denatures at 60oCusually optimum at 37oCthere is a characteristic increase in the

reaction rate for every 10oC increase before denaturation

Cofactorsmetals – transition metals (Zn++. Cu++ and

Fe++) – effective electophiles

InhibitorsCompetitive (compete for active site)Non-competitive (binds with enzyme at

place other than active site)Uncompetitive (binds with E-S complex)

Enzymes expressed in units that represent one of the following:

Increased concentration of one of the products substrate and coenzyme

The rate of change of any 3 unit is a measure of rate of reaction

The catalytic rate is proportional to its concentration at normal condition

MICHEALIS-MENTEN HYPOTHESIS k1 k3

E + S (ES) E + P

k2

where: k1 is rate constant for ES formation

k2 is rate constant for ES dissociation

k3 is rate constant for product formation and release from

active site

International Union of Biochemistry (IUB)Classify and name enzymes according to the type of

chemical reaction it catalyzesUsing the name of the substrate or group on which

the enzymes acts and added by suffix “-ase”Examples:

Urease hydrolyzes ureaAmylase metabolizes starch/amylumPhosphatase acting on phosphate esters

Biochemical functions, indicating substrate, class of reaction catalyzed designated by individual identification numbers

For clarity, the raction is also identified (examples are carbonic anhydrase, D-amino acid oxidase and succinic dehydrogenase)

Systemic name – nature of the reaction catalyzed is associated with unique numercal code designation

Of two parts: substrate(s) acted upon and a word ending with –ase indicating the reaction involved

Example: L-Lactate:NAD+ oxidoreductase

Trivial or practical name – may be identical to the systemic name but is often a simplification of it – useful in everyday work

Example: EC 1.1.1.27 L-Lactate:NAD+ oxidoreductase

lactate dehydrogenase

EC denotes Enzyme CommisionFirst number defines the class to which the enzyme

belongs

Assigned to 6 classes1. oxidoreductases2. transferases3. hydrolaseses4. lyases5. isomerases6. ligases

next two numbers indicate the subclass and sub-subclass to which the enzyme is assigned

Example: may be differentiated from the amino transferring subclass of the phosphate – transferring category or the ethanol acceptor sub-class from that accepting acyl group

The last number is the specific serial number given to the enzyme within its subclass

1. oxidoreductases - catalyzed oxidation-reduction reactions

REDUCTION (addition of hydrogen to a double bond)OXIDATION (removal of a hydrogen from a molecule to

leave a double bond)The hydrogen is transferred with the use of coenzyme

L-lactate:NAD+ oxidoreductase catalyzed pyruvate + NADH + H+ lactate + NAD+

subclasses: dehydrogenases, oxidases, oxygenases, reductases, peroxidases, and hydroxylases

2. transferases – catalyzed reactions that involve the transfer of groups from one molecule to another (amine or phosphate group)

ATP:creatine N-phosphotransferase (creatine kinase) involves

ATP + creatine ADP + creatine phosphate

trivial names include with prefix “trans”: transcarboxylases, transmethylases and transaminases

3. hydrolases – catalyze reactions in which the cleavage of bonds is accomplished by adding water

Amylase (cleavage of –C-O-C- bonds in starch)Lipase (breaks down triglycerides to form glycerol and free

fatty acids)Acid phosphatase and alkaline phosphatase (remove

phosphate group from a variety of molecules)

subclasses: esterases, phosphatases, peptidases

4. lyases – (lysis means “splitting”) catalyze reactions in which groups are

removed to form a double bond or are added to a double bond (C-C, C-S, and C-N bonds)

Aldolase (EC 4.1.2.13, D-fructose-1,6-biphosphate-D-glyceraldehyde-3-phosphate lyase) which cleaves the 6-carbon molecule fructose-1,6-diphosphate to produce two 3-carbon compounds: glyceraldehyde-3-phosphate and dihydroxyacetone phosphate

subclasses: decarboxylases, hydratases, dehydratases, deaminases, synthases

5. isomerases – (heterogenous group) catalyze several types of intramolecular rearrangements

Where it involves in the conversion of one isomer to another with examples of transformation will include the change of cis to trans; L-form to D-form; aldehyde to ketone

the reactions are generally reversible

Triose phosphate isomerase (EC 5.3.1.1, D-glyceraldehyde-3-phosphate ketol-isomerase)

In the glycolytic pathway, involves in the isomerization of glyceraldehyde-3-phosphate (aldehyde) to dihydroxyacetone phosphate (ketone)epimerases – catalyze the inversion of

assymmetric carbon atomsmutases – catalyze the intramolecular transfer of

functional group

6. ligases – catalyze bond formation between two substrate molecules forming a larger molecule

Important in the activation of amino acids – protein synthesisEnergy is always supplied by ATPAminoacyl-tRNA

1. Substrate Measurement This starts with a high substrate

concentration

2. Product Measurement This starts with zero initial product level This is more accurate

Endpoint analysis the reaction is initiated by addition of substrate

and is allowed to proceed for a period of timemeasurement of substrate or product is done at

the end of the reaction

Multipoint assaythis measures the change in the concentration of

the indicator substance at several intervals during the course of the assay

Kinetic assaythis involves measurement of change in

concentration as a function of timeclosely monitored at short intervalhas advantage over the end pointif the concentration of the substrate is sufficiently

high in comparison to enzyme then rate of reaction will be proportional to the concentration of enzyme

thus the amount of product formed in a given period of time would be proportional to the amount of active enzyme present, with all other factors remaining constant

Use of coupled reactionsenzymatic activity is measured by

coupling the activity with colorimetric reaction

the colored product is measured spectrophotometrically

1. HEMOLYSIS May cause falsely elevated enzyme

concentration

2. ANTICOAGULANTS Many anticoagulants cause adverse effects on

enzyme inhibiton, therefore, serum is preferred over plasma

3. LACTESCENT OR MILKY SERUM May result in variable absorbance readings in

spectrophotometry

Enzymes are stable at 6oC for at least 24 hours and at room temperature for lesser periods

For prolonged storage, use -20oC or lowerCK must be kept at -70oCLD4 and LD5 – liver isoenzymes are

inactivated at refrigerator temperature

That’s all folks… Have a great day ahead!