Download Enzymes I

Enzymes I
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Many drugs target the active site of enzymes
Enzyme deficiency is cause of many diseases
o Abnormal amino acid substitutions may have abnormal activity and cause disease (PRPP
synthetase and gout)
o An enzyme in a multi-enzyme pathway may be missing, causing the whole pathway to
fail (OTC deficiency and the urea cycle)
Catalysts that carry out biochemical reactions
Catalyst: a substance that changes the rate of a chemical reaction without affecting the position
of the equilibrium or being changed during the course of the reaction.
o Most biochemical reactions will not proceed at any rate in the absence of an enzyme.
o Enzymes increase the rates of biochemical reactions by 104 to 1010.
o Enzyme-catalyzed reactions proceed with very few or no side reactions. Chemical
catalysts usually catalyze a number of unwanted side reactions.
o Most enzyme-catalyzed reactions are fully reversible.
Enzymes have an active site
o Substrates bind in a cleft of the protein called the binding site, also known as the
catalytic or active site of the enzyme.
o Catalysis usually involves the actions of one or more amino acid residues with reactive
side chains (usually ser, cys, his, lys, glu, asp) that are specifically located near the
substrate binding site
o Cofactors: metal ions or coenzymes, assist enzymes during catalysis of reactions
o Highly specialized and selective (stereo specific) by tertiary structure
Enzyme Classes
o Oxidoreductases –add or remove one or more electrons from or to substrates.
 Oxidoreductases are frequently called “dehydrongenases” because their
oxidation reactions often remove two electrons and a proton from the oxidized
substrate
o Transferases –transfer a group from one substrate to another.
 includes kinases that transfer phosphate groups from ATP to substrates.
o Hydrolases – carry out the hydrolysis of substrates.
 includes phosphatases that remove phosphate groups from substrates.
o Lyases – carry out the cleavage of a molecule into parts
o Isomerases – rearrange a substrate into an isomeric form (i.e., D to L form)
o Ligases – join molecules together by condensation, usu. eliminating water.
o Isozymes – different enzyme that carry out the same reaction but with different
regulatory properties or in different locations within the organism (liver vs. heart, etc)
Energy changes in chemical reactions
o Free energy is a measure of the spontaneity of a reaction, i.e., which direction a reaction
is likely to proceed
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The change in Gibbs free energy (ΔGo’) for a reaction is the difference in energy between
the products and the substrates.
 ΔG < 0, the reaction proceeds in the forward direction spontaneously
enzyme catalysis changes the rate at which the equilibrium in the reaction is achieved
but does not change the position of the equilibrium.
 The equilibrium position is determined by the free energies of substrates and
products
 Rate of rxn depends on Ea, which is the energy to get to the transition state
 Enzymes increase the rate of rxn by decreasing the reaction Ea
Enzymes increase the rate of a rxn
o Binding Energy: interaction of S with enzyme active site provides binding energy,
bringing S closer in structure to the transition state. This lowers the activation energy
and increases the reaction rate.
o Proximity: Binding substrates increases their effective concentration within the active
site, thereby increasing the reaction rate.
o Induced Fit: Binding of S induces changes in the tertiary structure of E (conformational
changes) that in turn distort S toward the transition state.
o Orientation: geometry of S binding the active site will allow the reacting molecules to be
in the perfect orientation for reaction, thus increasing the reaction rate.
Stickase
o Goal is to break the substrate into pieces (which doesn’t happen spontaneously)
o S binds to E active site; favored conformation is to bend stick and form weak
interactions
 In process of bending, stick reaches transition state and breaks
 Two products released
Enzyme Kinetics
o Biochemical reactions almost always require an enzyme
o The enzyme concentration is much lower that that of substrate
o Thus, when substrate levels are high, the rate of the reaction depends on the
concentration of enzyme (i.e., the catalyst)
o Product formation is rate limiting step (covalent bonds broken/formed)
o In cells, level of P is usually low; P formation depends on rate of ES breakdown
o V=k[ES) where k is turnover number
Michaelis-Menton Equation v = Vmax {S} / Km + [S]
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Hyperbola; Vmax is when reach saturation/max binding
When [S]>>Km: v= Vmax
When [S] = Km: v= 0.5Vmax
When [S]<<Km: v = (Vmax[S])/Km
Hexokinase and glucokinase phosphorylate glucose; hemokinase has small Km therefore
high affinity and much quicker; opposite is true for glucokinase in liver
Multisubstrate reactions: If two substrates are required for rxn to procedd, both has influence
on curve
Serine Proteases
o a family of enzymes that hydrolyze specific peptide bonds in proteins (i.e., splits the
protein into smaller polypeptides)
o Each enzyme in the family hydrolyzes amide bonds adjacent to specific amino acid
residues.
o Main area of function: Digestion
o Serine proteases are synthesized in the pancreas as pro-enzymes or zymogens that are
inactive.
 The enzymes are activated after they are released from the pancreas.
o Protease specificity
 protease specificity is determined by a substrate-binding pocket
 Chymotrypsin: a hydrophobic binding pocket that favors aromatic residues and
large non-polar residues
 Trypsin: a deep binding pocket with a negatively charged residue (glu) at the
bottom that favors the binding of lys and arg
 elastase has a very small binding pocket that favors binding of ala or gly
residues.
Enzyme Catalysis: Chymotrypsin
o Cleaves bonds at certain locations, known by how it fits into active site (tyr residue)
o The “catalytic triad” of residues cooperate to make the Ser-OH a strong nucleophile,
which attacks the peptide carbonyl bond
o Transition state stabilizes
 Makes easier to form, then will break down to more stable products when
cleaved
 Activated water breaks the bond
 Peptide piece, first product, is released (fragment remains bound to
enzyme)
 Hydrolysis breaks ES bond
 Enzyme interacts with water, making strong nucleophile
o Regeneration of enzyme catalyst in order to leave again
Aspartyl Protease
o activation of water for nucleophilic attack, no enzyme-bound intermediates,
rearrangement of tetrahedral carbon intermediate leads to cleavage of peptide bond
HIV Protease
o HIV mRNA leads to synthesis of poly protein
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Proteolytic cleavage by HIV protease (an aspartyl protease) produces the individual
proteins the virus needs for growth and reproduction
HIV Protease Inhibitors slow spread of disease