Enzymology

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Enzymology

Enzymology

Deals with physical and chemical properties, function and clinical correlation of enzymes.

Enzyme

Found within the cell (intracellular). Biologic proteins that catalyze the biochemical reactions of organic reactants. Not consumed or altered.

Low

Enzyme is _____ in plasma

Functions of Enzymes

• Hydration of CO2 (Respiration)

• Nerve Induction

• Muscle Contraction

• Nutrient Degradation

• Growth and Reproduction

• Energy storage and Use

Active site

Waterless cavity of enzymes where substrates binds and undergo chemical reaction. Specific binding of substrate in the enzyme.

Allosteric site

Waterless cavity of enzymes other than active site that binds effector or regulatory molecule.

Substrates

Substances acted upon by enzymes. Specific for each enzyme.

Cofactors

Non-protein entities added to enzyme substrate complexes to enhance enzymatic activities.

Coenzyme

Often used in oxido-reductase activity. also called “Secondary Substrate.”

Activator

Usually a metallic ion added to enhance enzyme substrate binding. It enhances the active site conformation to allow binding.

Isoenzyme

Enzymes with similar activity but differ in physical, biochemical, and immunological activities.

Apoenzyme

Protein portion of enzyme. Loses its function when it undergoes protein denaturation.

Holoenzyme

Active substance formed by the combination of coenzyme and apoenzyme.

Prosthetic group

Coenzyme that is tightly bound to a enzyme.

Systematic name

Defines the substance acted upon enzyme and reaction catalyze and coenzyme involved. ALso known as the “long name of enzyme.”

Recommended name

Trivia or usable name.

EC numerical code

Enzyme commission contains 4 digits.

Enzyme class

1st digit of EC

Enzyme subclass

2nd digit of EC

Enzyme sub sub class

3rd digit of EC

Specific serial number for Enzyme sub sub class

4th digit of EC

Oxidoreductases

Catalyze redox reaction between 2 substrates.

Transferases

Catalyze a transfer of a group other than Hydrogen.

Hydrolases

Catalyze hydrolysis of various chemical bond

Lyases

Catalyze the removal of groups from substrates without hydrolysis. Product contains double bond.

Isomerases

Catalyze the interconversion of optical, geometric or positional form.

Ligases

Catalyze the joining of 2 substrate molecules coupled with the breaking of thiophosphate bond.

Catalytic Mechanism of enzymes

The lowering the activation energy level that the reactants must reach. A reactant must reach a certain activation energy level for a reaction to occur. If the reactant reaches the level immediately, there is faster product formation.

enzyme-substrate complex

When an enzyme and substrate perform, it will form an ____________________. The substrate will undergo transformation depending on the type of enzyme that acts upon it, while the enzyme remains unchanged.

Absolute Specificity

Enzymes combine with a specific substrate and catalyze only one corresponding reaction.

eg. Lactase – will only act upon lactose → glucose + galactose.

Group Specificity

Enzymes combine with all substrates containing a particular chemical group.

eg. Kinase will act on all substrates that contain a phosphate group.

Bond Specificity

Enzymes combine w/ substrates w/ a specific chemical bond.

eg. Lipase – will act on all substrates containing an ester bond

Stereoisomeric Specificity

Enzymes combine w/ only 1 optical isomer.

Factors Influencing Enzymatic Ractions

• Substrate concentration

• Enzyme concentration

• pH

• Temperature

• Cofactors

• Inhibitors

• Storage

• Hemolysis

• Milky / Fatty / lactescence

Substrate concentration

Directly proportional w/ enzyme activity. When more substrate is added, there is higher enzymatic action.

There is a limitation to which an enzyme can catalyze a reaction.

When all substrates had already attached to the active site of an enzyme, addition of any more substrate will no longer cause any enzymatic activity – ”saturation kinetics”

provided that there are available active sites the substrates can bind to.

1st order kinetics

Reaction rate is proportional to the substrate concentration.

*the rate of a ____ order kinetics DEPENDS on the substrate concentration.

zero order kinetics

Only a fixed number of substrates is allowed to attach to the active site, hence allowed to undergo interconversion / transformation.

*the rate of _____ order kinetics DOES NOT DEPEND on the substrate concentration because there is already a fixed or constant amount of substrate allowed to undergo a reaction.

Enzyme concentration

Directly proportional to the substrate concentration.

More enzyme = higher activity

pH

Enzyme activity occurs at a pH near plasma pH {7.35 - 7.45},

7-8 pH.

When pH is too acidic/ alkaline – promotes protein

denaturation. The apoenzyme loses its enzymatic

activity.

Temperature

As temperature increases, the enzymatic activity increases.

For every 10 °C increase in temperature for enzyme

incubation, there is a two-fold increase in enzyme activity –

“Q10“.

Competitive Inhibitor

physically binds to the active site of the enzyme, competing w/ the substrate. Affects the velocity / speed of chemical reaction that promotes the conversion of substrate to product.

Noncompetitive Inhibitor

Binds to the cavity other than the active site of the enzyme {allosteric site}.

Uncompetitive Inhibitor

Binds to the enzyme-substrate complex.

Storage

Freezer temperature; -18℃ or colder. Enzymes can be maintained for an indefinite period of time.

LDH – requires higher temp for storage: room temp must be maintained between 20-24℃

substrate & coenzyme – refrigerator temp must be maintained b/w 2-8℃

Once

You can only thaw / defrost a serum sample _____. You cannot “refreeze” a serum sample. Multiple refreezing-thawing procedures of serum destroy proteins, including enzymes.

Hemolysis

Hemolyse sample, RBCs also have enzymes Liberation of enzymes from hemolyzed RBCs → false increase enzymatic activity.

Milky / Fatty / lactescence

Fatty constituents of serum can interfere w/ substrate-enzyme binding or interaction → false dec enzyme activity.

INCREASE in product concentration

There is substrate conversion catalyzed by

enzymes – more substrates = more enzyme activity = more product produced

DECREASE in substrate concentration

As more product is formed, more substrate was converted.

DECREASE in coenzyme concentration

(NADH) reduced –> increased absorbance.

INCREASE in altered coenzyme conc’n

(NAD) oxidized – decreased absorbance

Measurement of catalytic activity is

• DEPENDENT on enzyme concentration

• Always performed in ZERO-order kinetics

• Performed during the LINEAR phase reaction

Fixed-Time Enzyme Activity Measuring

Direct / straight-forward.

Two reactants (enzyme present in serum + substrate/reagent) are

combined.

● There is only one incubation period and only one

measurement obtained.

Continuous Monitoring Enzyme Activity Measuring

Multiple enzyme measurements

● Done as specific intervals

● (1) The reactants are combined; (2) incubate; (3)

Measure the absorbance; – 30-50 sec time interval

(4) – 2nd measurement – incubate for 60 sec; (5)

Measure; (6) Incubate; (7) Measure

the progression of enzyme activity is monitored

deviation of zero kinetics can also be observed.

IU (EC)

International Unit; Enzyme Commission

The amount of enzyme that will catalyze the reaction of 1 umol substrate/min.

Kat (SI)

Katal

The amount of enzyme that will catalyze the reaction of 1 mol of substrate/sec.

Lock & Key Theory

Theorized by Emil Fisher.

The shape of the key (substrate) must fit into the lock (active site of enzyme).

Induced Fit Theory

Theorized by Kochland.

Based on the substrate binding to the active site of the enzyme.

The active site of an enzyme will conform to the shape of the

substrate upon binding.