MLAB 2401: Clinical ChemistryKeri Brophy-Martinez

Enzymes: Overview

Enzymes

• Functional proteins that catalyse biological reactions

• Involved in all essential body reactions• Found in all body tissues– Seen in serum following cellular injury or from

degraded cells

• Decrease the amount of free energy needed to activate a specific reaction

General Properties of Enzymes

• Not altered or consumed during reaction• Reusable• Accelerate speed of reactions

General Properties of Enzymes

• Holoenzyme– Functional unit– Consists of:• Apoenzyme• Cofactor/coenzyme

• Proenzyme/zymogen– Inactive enzyme

Holoenzyme

General Properties of Enzymes

• Role– Increase reaction rates while not being consumed

or altered

Enzyme

– Substrate Product

Definitions and Related Terms

• Active site– Specific area of the

enzyme structure that participates in the reaction(s)/interacts with the substrate

Definitions and Related Terms

• Allosteric site– Non-active site– May interact with other

substances resulting in overall enzyme shape change

Definitions and Related Terms

• Isoenzymes– Structurally different enzymes that catalyze the

same reaction• Multi molecular form• Similar catalytic activity• Differing biochemical or immunological characteristics• Can detect by different electrophoresis patterns,

absorption patterns, or reaction with specific antibodies

Definitions and Related Terms

• Cofactor– Non-protein substances required for normal

enzyme activity– Types• Activator: inorganic material such as minerals

– (Ca 2+, Fe2+)

• Co-enzymes: organic in nature– (ATP, ADP, nicotinamide)

Enzyme Kinetics

• Reactions occur spontaneously if energy is available

• Enzymes lower the activation energy for the chemical reactions

Enzyme Kinetics

• Activation energy– Excess energy that

raises all molecules at a certain temperature to the activation state

Enzyme Kinetics

• Basic reaction– S + E ES E + P

– Where• S= substrate

– Substance on which the enzyme acts• E= Enzyme• ES= enzyme-substrate product

– Physical binding of a substrate to the active site of enzyme• P= Product

Enzyme Kinetics & Specificity

• Enzymes differ in their ability to react with different substrates– Absolute specificity

• Enzyme combines with only one substrate and catalyzes one reaction

– Group specificity• Combine with all substrates containing a specific chemical group

– Bond specificity• Enzymes specific to certain chemical bonds

– Stereoisomerism• Enzymes that mainly combine with only one isomer of a particular

compound

Michaelis-Menten

• Relationship of the reaction velocity/rate to the substrate concentration

• The Michealis-Menten Constant (Km)• The substrate

concentration in moles per liter when the initial velocity is ½ V max.

Michaelis-Menten Curve

Michaelis-Menten

• First order kinetics– Rate is directly

proportional to substrate concentration

• Zero order kinetics– Plateau is reached– depends only on enzyme

concentration

Michaelis-Menten• Equation used to distinguish different kinds of

inhibition

• Where– V0: velocity/rate of enzymatic activity– Vmax: The maximal rate of reaction when the enzyme is

saturated– Km: (constant)the substrate concentration that

produces ½ of the maximal velocity– S: substrate concentration

Lineweaver-Burk Plot

• Adaptation of Michaelis-Menten equation

• Yields a straight line

Influencing Factors on Enzymatic Reactions

• Substrate Concentration• Enzyme Concentration

– The higher the enzyme level, the faster the reaction• pH

– Most reaction occur in range of 7.0-8.0– Changes in pH can denature an enzyme

• Temperature– Most reactions performed at 37 o C– Increasing temp increases rate of reaction– Avoid high/low temps due to denaturation of enzyme

• Cofactors– Influence the rate of reaction

• Inhibitors– Presence can interfere with a reaction can be reversible or irreversible

Types of Inhibition

• Competitive– Any substance that competes with the substrate for

the active binding sites on the substrate– Reversible

• Non-competitive– Any substance that binds to an allosteric site

• Uncompetitive– Inhibitors bind to the ES complex– No product produced

Noncompetitive Inhibition

Irreversible Inhibition

Competitive Inhibition

Types of Inhibition

Competitive Noncompetitive Uncompetitve

Enzyme Nomenclature

• Historical– ID of individual enzymes was made using the

name of the substrate that the enzyme acted upon and adding “ase” as the suffix

– Modifications were often made to clarify the reaction

– International Union of Biochemistry (IUB) in 1955 appointed a commission to study and make recommendations on nomenclature for standardization

Enzyme Nomenclature: IUB• Components

– Systematic name• Describes the nature of the reaction catalyzed• Example: alpha 1,4-glucagon-4-gluconohydrolase

– Recommended name• Working or practical name• Example: amylase

– Numerical code• First digit places enzyme in a class• Second and third digit represent subclass(s) of the enzyme• Fourth digit specific serial number in a subclass• Example: 3.2.1.1

Enzyme Nomenclature: IUB

• Standard Abbreviated name– Accompanies recommended name– Example: AMS

• Common Abbreviated name– Example: AMY

Enzyme Classification: General

• Plasma vs. non-plasma specific enzymes– Plasma specific enzymes have a very definite/

specific function in the plasma• Plasma is the normal site of action• Concentration in plasma is greater than in most tissues• Often liver synthesized• Examples: plasmin, thrombin

Enzyme Classification: General

– Non-plasma specific enzymes have no known physiological function in the plasma• Some are secreted in the plasma• Increased number of this type seen with cell disruption

or death

Enzyme Classification

• Six classes– Oxidoreductases

• Involved in oxidation-reduction reactions• Examples: LDH, G6PD

– Transferases• Transfer functional groups from one substrate to another• Examples: AST, ALT

– Hydrolases• Catalyze the hydrolysis of various bonds• Examples: acid phophatase, lipase

Enzyme Classification

– Lyases• Catalyze removal of groups from substrates without

hydrolysis, product has double bonds• Examples: aldolase, decarboxylase

– Isomerases• Involved in molecular rearrangements• Examples: glucose phosphate isomerase

– Ligases• Catabolism reactions with cleavage of ATP• Example: GSH

References

• Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins.

• http://regentsprep.org/Regents/biology/units/homeostasis/processes.cfm

• http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/fg9.html

• Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .