BIOCHEMISTRY: TCA CYCLE, GLUCONEOGENESIS, ENZYME KINETICS

Enzymes

· Enzyme naming nomenclature

· Systematic Name assigned by international union of biochemistry and molecular biology (IUBMB)

· Enzymes divided into six major classes

· Recommended Name, two derivations

· Substrate + “ase”

· e.g., Urase

· Reaction catalyzed + “ase”

· e.g., Lactate dehydrogenase

· Trivial/Historical Name

· Gives little or no operational information

· e.g., Pepsin, Trypsin

· Catalysts

· Types

· Metals

· Protein enzymes

· Ribozymes

· Protein enzyme properties

· Active Sites

· Binding site for substrate

· Forms [ES] = Enzyme substrate complex

· Enzyme site in which reaction takes place

· Contain AA side chains/ functional groups that help catalyze reaction

· Catalytic efficiency

· Can increase reaction velocity by 1x103 to 1x108 compared to uncatalyzed reaction

· Specificity

· Catalyst is specific when it utilizes a few select substrates

· The ability to discriminate between substrate & some other competing molecule

· Cofactors

· Additional chemical groups or components that may be required for enzyme activity

· Definitions

· Holoenzyme

· Hol-

· word-forming element meaning "whole, entire, complete," from Greek holos

· Refers to catalytically active enzyme with cofactors

· Apoprotein (apoenzyme)

· Enzyme without cofactors

· Cofactors

· Inorganic ion

· e.g., Fe2+, Zn2+

· Coenzymes

· Organic or metalloorganic molecule

· Many are derivatives of vitamins

· Prosthetic group

· Tightly or covalently bound cofactor

· e.g., Heme

· Theory of operation

· Accelerates the completion of a reaction

· Does not change reaction equilibrium

· Energy changes during reaction

· Energy barrier of reaction = free energy of activation

· Energy difference between reactants and transition state (highest energy intermediate), T*

· Reaction rate determined by reaction step with the highest energy of activation

· Called rate limiting step

· E + S ↔ ES ↔ T* ↔ EP ↔ E + P

· Free energy of activation, ΔG‡

· Energy difference between reactants and transition state

· Higher ΔG‡ the slower the uncatalyzed reaction rate

· Enzymes increase rate of reaction

· Enzymes reduce ΔG‡

· Increases reaction rate

· Greater proportion molecules can reach transition state

· Enzymes do not alter free energies of reactants or products

· Enzymes do not alter reaction equilibrium

· Active site chemistry, mechanisms which facilitate conversion of substrate, S

· Active site binding forces S to assume geometry of transition state

· Stabilizes transition state for reaction

· AA side chains may function as catalytic groups

· Reaction velocity, V

· Usually expressed as μmol of product formed / min

· Variables affecting velocity

· Reactant concentration

· Rate increases with S concentration, [S], until Vmax approximately attained

· Kinetics curve

· Plot of initial velocity, Vo, vs. substrate concentration

· Hyperbolic shape

· Consistent with Michaelis-Menton kinetics

· Sigmoidal curve

· Allosteric enzymes

· e.g., oxygen dissociation curve of hemoglobin

· Temperature

· Velocity increases with temperature until peak reached

· The higher the temp, the greater the proportion of molecules of sufficient energy to overcome barrier

· Decrease in velocity after peak

· Temperature-induced denaturation of enzyme

· pH

· Changes may increase or decrease velocity

· Ionization state of reactants & enzymes change altering velocity

· Optimum pH for reaction velocity varies with enzyme

· e.g., Pepsin requires lower pH

· e.g., Alkaline phosphatase requires higher pH

· Enzymes which obey the Michaelis-Menten equation

· Reaction model:

·

·

·

·

· Where,

· E is enzyme

· S is substrate

· ES is the enzyme-substrate complex

· P is product

· k1, k-1, k2 are unimolecular rate constants

· Michaelis-Menten Equation

·

·

·

·

· Where,

· Km = Michaelis constant (M)

· Vo = Initial rxn velocity (μmol·min-1)

· Vmax = Maximum rxn velocity (μmol·min-1)

· [S] = Substrate concentration (Moles)

· Assumptions

· [S]>>[E]

· Substrate consumed is insignificant

· [ES] constant

· Rate of ES formation = ES breakdown

· System achieves steady state

· V0 measured at steady state

· Initial velocity used to analyze enzyme reactions

· Measured immediately after steady state achieved

· Called “Steady-state kinetics”

· Early in reaction [P] is negligible

· P -> S can be ignored, thus:

· V0 determined by breakdown rate of ES

· V0 = f([S]) = rectangular hyperbola

· Interpretation of variables

· Km = Michaelis constant

· Related to affinity of enzyme for substrate

· Practical definition

· Equal to [S] at which Vo = ½Vmax

· Lower Km implies higher affinity

· Higher Km implies lower affinity

· Reaction order

· When [S]<< Km

· V0([S]) is first order or linear

· When [S] >> Km

· V0([S]) = Vmax

· V0([S]) is zero order or constant

· Lineweaver-Burke plot (double-reciprocal plot)

·

·

· FORM: y = mx + b

· X axis intercept = -1/Km

· Y axis intercept = 1/ Vmax

· Enzyme inhibitors

· Decrease velocity of enzymatically catalyzed reactions

· Mechanisms of inhibition

· Competitive inhibition

· Inhibitor binds reversibly to substrate binding site

· Increases apparent Km

· Greater [S] required to achieve Vmax

· Vmax unchanged

· e.g., Statin drugs – reduce cholesterol production

· Noncompetitive inhibition

· Inhibitor binds to site other than the substrate binding site

· Decreases Vmax

· No increase with increase in [S]

· Km does not change

· Does not alter affinity of S for E

· Example: ACE inhibitors decrease blood pressure

· Cellular regulation of enzyme activity

· Most cellular enzymes operate in an environment where with [S] ≈ Km

· Changes in [S] change rate of reaction

· Allosteric regulation

· Some enzymes regulated by allosteric effectors (modifiers)

· Bind noncovalently to site other than active site

· Effectors may modify substrate affinity, Vmax, or both

· Negative effectors – inhibit activity

· Positive effectors – enhance activity

· Example:

· Product of reaction can inhibit enzyme

· Example of negative feedback

· Regulation by covalent modification

· Phosphorylation may increase or decrease activity

· Phosphoprotein phosphatases cleave phosphate groups

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