MLAB 2401: Clinical Chemistry Keri 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
Where
S= substrate
Substance on which the enzyme acts

ES

E+P

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 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 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/p rocesses.cfm http://student.ccbcmd.edu/~gkaiser/biotutorials/proteins/fg9 .html Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .