MLAB 2401: Clinical Chemistry Keri Brophy-Martinez

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MLAB 2401 Clinical ChemistryKeri Brophy-Martinez

• Enzymes Overview

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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

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General Properties of Enzymes

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

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General Properties of Enzymes

• Holoenzyme
• Functional unit
• Consists of
• Apoenzyme
• Cofactor/coenzyme
• Proenzyme/zymogen
• Inactive enzyme

Holoenzyme
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General Properties of Enzymes

• Role
• Increase reaction rates while not being consumed
  or altered
• Enzyme
• Substrate Product

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Definitions and Related Terms

• Active site
• Specific area of the enzyme structure that
  participates in the reaction(s)/interacts with
  the substrate

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Definitions and Related Terms

• Allosteric site
• Non-active site
• May interact with other substances resulting in
  overall enzyme shape change

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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

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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)

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Enzyme Kinetics

• Reactions occur spontaneously if energy is
  available
• Enzymes lower the activation energy for the
  chemical reactions

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Enzyme Kinetics

• Activation energy
• Excess energy that raises all molecules at a
  certain temperature to the activation state

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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

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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

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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
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Michaelis-Menten

• First order kinetics
• Rate is directly proportional to substrate
  concentration
• Zero order kinetics
• Plateau is reached
• depends only on enzyme concentration

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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

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Lineweaver-Burk Plot

• Adaptation of Michaelis-Menten equation
• Yields a straight line

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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

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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

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Noncompetitive Inhibition
Irreversible Inhibition
Competitive Inhibition
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Types of Inhibition
Uncompetitve
Noncompetitive
Competitive
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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

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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

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Enzyme Nomenclature IUB

• Standard Abbreviated name
• Accompanies recommended name
• Example AMS
• Common Abbreviated name
• Example AMY

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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

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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

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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

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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

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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/homeo
  stasis/processes.cfm
• http//student.ccbcmd.edu/gkaiser/biotutorials/pr
  oteins/fg9.html
• Sunheimer, R., Graves, L. (2010). Clinical
  Laboratory Chemistry. Upper Saddle River Pearson
  .