bio chem 460 quarter 3

enzyme

• biological catalysts that increase reaction rates without being consumed.

• Required in small quantities for cellular reactions.

• Mostly globular proteins.

ribozymes/ribosomal RNA

also exhibit catalytic activity

apoenzyme

• Inactive protein component alone.

Cofactor

• Non-protein helper molecule (metal ion or organic molecule).

Coenzyme

 Organic cofactor (often vitamin-derived)

Holoenzyme

• Complete, active enzyme (apoenzyme + cofactor).

Advantages of biocatalysts over inorganic catalysts (specificity)

• Enzymes selectively bind substrates → fewer side products.

Advantages of biocatalysts over inorganic catalysts (mild conditions)

 Operate at pH ~7, 37°C, 1 atm

Advantages of biocatalysts over inorganic catalysts (regulation)

• Activity controlled by allosteric and covalent mechanisms.

Advantages of biocatalysts over inorganic catalysts (high reaction rates)

• Increase rates by factors of 10⁶–10¹².

substrate selectivity

• Determined by the structure of the binding pocket.

• Ensures only the correct substrate binds and reacts. Incorrect substrates either do not bind or fail to react.

example of substrate selectivity

 Phenylalanine hydroxylase only binds phenylalanine, not tyrosine.

binding site

• Region that holds substrate via weak interactions.

active site

• Location of catalysis; contains critical residues.

enzymes and reaction energetics

• Enzymes lower activation energy (ΔG‡) but do not change ΔG′° or Keq.

• They do not affect equilibrium — only the rate at which equilibrium is reached.

• Spontaneous (ΔG < 0) reactions still require enzymes due to kinetic barriers.

delta G ‘ degree

 Standard free energy change — determines thermodynamic favorability.

delta G t

• Activation energy — determines reaction rate.

Transition state

• Unstable, high-energy configuration at reaction peak.

Intermediate

• More stable species between reaction steps.

ES (Enzyme–Substrate) reaction complex

• Substrate bound to enzyme.

EP (Enzyme–Product) reaction complex

Product bound before release

Enzyme catalysis equation

• E + S ⇌ ES → EP → E + P.

Catalytic Rate Enhancement

• Enzymes accelerate reactions by 10⁶–10¹²×.

Catalytic Rate Enhancement example

Chymotrypsin increases peptide bond cleavage rate by 10⁹×.

How Enzymes Lower Activation Energy

• Organize reactive groups → proper proximity & orientation.

• Restrict substrate motion → reduce entropy cost.

• Destabilize substrate bonds (induced strain).

• Stabilize transition state through weak interactions (binding energy).

• Replace unfavorable solvation shell with enzyme interactions.

Entropy

 Random movement reduces reaction likelihood.

Solvation shell

Hydrogen-bonded water must be removed.

Substrate distortion

 Bonds must bend/stretch.

Alignment

Catalytic groups must orient correctly.

Transition state binding

• Enzyme active sites are complementary to transition states, not substrates.

Linus Pauling

•  proposed transition-state stabilization model.

• Stronger binding to transition state → lower ΔG‡ → faster rate.

Transition-state analogs

act as potent inhibitors.

Proximity and orientation model

• Binding brings reactive groups into close proximity and proper orientation.

Thomas C. Bruice

• experiments on ester–carboxylate reactions.

Acid–Base Catalysis

• Proton donation/acceptance stabilizes intermediates.

Covalent Catalysis

Temporary covalent bond forms between enzyme & substrate.

Metal Ion Catalysis

• Metals stabilize negative charges or participate in redox reactions.

His, Asp, Glu, Lys, Tyr, Cys

Amino acids commonly in Acid–Base Catalysis

Ser, Lys, Cys, Asp, His.

Amino acids commonly in Covalent Catalysis

Oxidoreductases

Redox reactions

Transferases

Transfer functional groups

Hydrolases

Hydrolyze bonds.

Lyases

Add/remove groups to form double bonds.

Isomerases

Rearrange atoms within a molecule

Ligases

Form bonds using ATP hydrolysis.

protease function

• Cleave peptide bonds in proteins.

Classes of protease

Serine, Cysteine, Aspartyl, Metalloproteases.

importance of protease

• Digestion, zymogen activation, viral maturation.

Chymotrypsin cleaves

• Peptide bonds next to aromatic residues (Phe, Tyr, Trp).

Chymotrypsin Catalytic Triad

Ser195, His57, Asp102 → hydrogen-bonding network.

Chymotrypsin mechanism steps

1. Substrate binds hydrophobic pocket.

2. Nucleophilic attack by Ser195 → tetrahedral intermediate.

3. Peptide bond cleavage → acyl–enzyme intermediate.

4. Water enters, deprotonated by His57.

5. Hydrolysis regenerates free enzyme.

Chymotrypsin stabilization

• Oxyanion hole stabilizes transition state.

Chymotrypsin rate enhancement

• ~10⁹-fold faster than uncatalyzed reaction.

HIV Protease function

• Cleaves viral polyproteins into functional units.

HIV Protease specificity

Between Phe and Pro residues.

HIV Protease inhibitors

• Contain –OH mimicking tetrahedral intermediate; block enzyme activity.

HIV Protease importance

Critical drug target in HIV treatment

Chymotrypsin type

Serine protease.

Lysozyme substrate

•  Peptidoglycan (bacterial cell wall).

Lysozyme cleaves

• β(1→4) bond between N-acetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc).

Lysozyme active site residues

• Glu35 (acid/base), Asp52 (nucleophile).

Lysozyme mechanism

• Two successive SN2 steps; involves covalent glycosyl–enzyme intermediate.

Enolase type

Metal ion catalysis enzyme.

Enolase function

• Catalyzes dehydration in glycolysis.

Enloase cofactor

Two Mg²⁺ ions stabilize oxyanions and assist proton removal from C-2.

Hexokinase function

• Phosphorylates glucose → glucose-6-phosphate.

Hexokinase mechanism

• Induced fit — conformational change aligns active site residues.

Hexokinase importance

• Demonstrates enzyme flexibility and substrate specificity.

Penicillin mechanism

Irreversible inhibitor of bacterial transpeptidase.

Penicillin effect

• Blocks cell-wall cross-linking → bacterial lysis.

Penicillin resistance

 β-lactamases hydrolyze penicillin’s β-lactam ring.

Basic reaction pathway

• E + S ⇌ ES → E + P

steady state assumption

• [ES] remains constant once initial equilibrium is reached.

Michaelis–Menten Equation

v=Vmax[S]Km+[S]v=Km​+[S]Vmax​[S]​

Vmax

• Maximum velocity (enzyme saturated).

Km

• Substrate concentration at ½Vmax (approx. substrate affinity).

Kcat

• Turnover number — number of substrate molecules converted per enzyme per second.

Kcat/Km

• Specificity constant — measure of catalytic efficiency.

Effect of low substrate concentration

 v ≈ (Vmax/Km)[S] (linear increase).

Effect of high subtrate concentration

• v ≈ Vmax (enzyme saturated).

Enzyme efficiency

• Limited by substrate diffusion rate (~10⁸–10⁹ M⁻¹s⁻¹).

Example of enzyme efficiency

• Catalase = high kcat, high Km; Acetylcholinesterase = low Km, high kcat.

Lineweaver–Burk (Double-Reciprocal) Plot

1v=KmVmax1[S]+1Vmaxv1​=Vmax​Km​​[S]1​+Vmax​1​

• Straight line form of Michaelis–Menten.

• Useful for determining kinetic parameters and inhibition type.

Slope and Y intercept of Lineweaver-Burk (double reciprocal) plot

• Slope = Km/Vmax; Y-intercept = 1/Vmax; X-intercept = –1/Km.

Sequential (Single-Displacement) two substrate reaction

• Both substrates bind before any product forms.

• May be ordered or random.

• LB plots: Lines intersect (indicating ternary complex).

Ping-Pong (Double-Displacement) two substrate reaction

• One substrate binds, product released before second binds.

• Enzyme alternates between modified/unmodified forms.

• LB plots: Parallel lines.

Irreversible Inhibition

• Inhibitor covalently modifies or destroys enzyme.

• One inhibitor molecule permanently inactivates one enzyme.

• Often toxins or drugs (e.g., penicillin).

Reversible inhibition

• Noncovalent, dissociable binding of inhibitor.

• Inhibitors resemble substrate or product analogs.

Competitive inhibition

Inhibitor competes with substrate, Vmax is unchanged, Km increases, in LB plot lines intersect at Y axis

Uncompetitive inhibition

Inhibitor binds only ES complex, Vmax decreases, Km decreases, in LB plot lines are parallel

Mixed (noncompetitive) inhibition

Inhibitor binds E or ES, Vmax decreases, Km varies, in LB plot lines intersect left of y axis

Allosteric Regulation

• Effector binds at non-active site → conformational change.

• Positive (activator): Increases enzyme activity.

• Negative (inhibitor): Decreases enzyme activity.

• Produces sigmoidal curve (cooperative binding).

Allosteric Regulation example

• ATCase inhibited by CTP, activated by ATP.

Covalent Modifications

Chemical modification alters enzyme activity.

Reversible types of covalent modifications

• Phosphorylation, adenylation, acetylation, methylation, ubiquitination.

Irreversible types of covalent modifications

• Proteolytic cleavage (zymogen activation).

Zymogens

Inactive enzyme precursors activated by cleavage Used in digestive and clotting cascades. Activation is irreversible.

Zymogen example

Trypsinogen → Trypsin, Fibrinogen → Fibrin.

Carbohydrates

• Formula: (CH₂O)n. Derived from CO₂ and H₂O via photosynthesis.

• Range in size from glyceraldehyde (MW 90) to amylopectin (MW > 200 million).

Carbohydrates function

• Energy source, storage, structure, communication.