unit 2 biochem

Myoglobin and Hemoglobin

• Homologous proteins with similar primary sequences and functions.
  • Both bind oxygen.
  • Both contain a heme ring stabilized by histidine.
• Hemoglobin travels in the blood inside red blood cells to deliver oxygen to tissues.
• Myoglobin remains in the heart and skeletal muscle cells to bind oxygen released by hemoglobin.
• Tertiary and quaternary structures are stabilized by hydrophobic interaction, hydrogen bonds, and salt bonds.
• Apoprotein: A protein missing its ligand or ligands (e.g., hemoglobin lacking heme).
• Holoprotein: A protein with its ligand, enabling it to function (e.g., hemoglobin bound to heme).

Myoglobin

• Monomer with 8 α-helices linked by α-turns.
• Has a hydrophobic pocket containing heme with a ferrous iron atom ($Fe^{+2}$) at its center for oxygen binding.
• $Fe^{2+}$ is always bound to a histidine R-group of the α-helix, stabilizing the reduced state of iron when it binds to oxygen.
• Heme is tightly bound to the globin, termed a prosthetic group.
• Myoglobin binding follows a hyperbolic curve.

Hemoglobin (Hb)

• Heterotetramer composed of 2 α and 2 β subunits.
• Each subunit has its own heme; hemoglobin can bind 4 oxygen molecules.
• Cooperativity: Alteration of an enzyme (like hemoglobin) consisting of several subunits by the substrate.
  • When $O_2$ binds to the $Fe^{2+}$ at one of the Hb binding sites, it pulls on the histidine, which pulls on the α-helix, changing the conformation of the globin slightly. This movement changes the conformation of the other three chains in the Hb.
  • When oxygen binds to one heme, the other hemes are more likely to bind a second molecule of oxygen.
• Hemoglobin does not bind oxygen as strongly as myoglobin, illustrated in the sigmoidal binding curve.
• When oxygen is released from hemoglobin, the loss of one molecule of oxygen facilitates the loss of additional oxygen molecules (reverse of positive cooperativity).

Enzyme Kinetics

Michaelis-Menten Equation

$v<em>i = (V</em>{max} [S]) / (K_m + [S])$

• $v_i$ = initial velocity (initial rate of the reaction at a certain substrate concentration)
• $V_{max}$ = maximal velocity (rate) a reaction can achieve at an infinite concentration of substrate.
• $K_m$ = Michaelis constant; substrate concentration at which the reaction rate is at half-maximum, indicating the substrate's affinity for the enzyme.
  • A small $K<em>m$ indicates high affinity, with the rate approaching $V</em>{max}$ at lower substrate concentrations.
  • $K<em>m$ and $v</em>i$ are inversely related.
• $[S]$ = substrate concentration (the rate of the reaction is dependent on the amount of substrate).
• $\Delta G$ - The presence of an enzyme has no impact on overall energy

Factors Influencing Reaction Rates

• Temperature
• Hydrogen ion concentration
• Oxidation: the loss of electrons
• Reduction: the gain of electrons

Inhibitors

Competitive Inhibitor

• Binds in the active site.
  • $K_m$ increases: Substrate concentration must increase to compete with the inhibitor.
  • $V_{max}$ remains the same: High substrate concentration reduces the chance of inhibitor binding.
  • Slows enzyme reaction at any substrate concentration due to active site competition.

Free Energy in a System

• First law of thermodynamics: total energy remains constant.
• Second law of thermodynamics: In all spontaneous reactions, entropy always increases when both the system and environment are taken into account.
• Entropy: disorder or randomness of a system
• Enthalpy: Internal energy of a system
  • If heat is given off enthalpy decreases; if heat is absorbed enthalpy increases
• Endergonic: A reaction that requires the absorption of energy (e.g. energy must be added to the reaction for it to proceeded)
• Exergonic: A reaction that gives off energy to its surroundings

ΔG - Change in Free Energy

• If positive (endergonic): reaction requires energy to proceed (e.g., anabolic).
• If negative (exergonic): the reaction is spontaneous (e.g., catabolic).
• Coupling reactions allows for a system to organize reactions, using energy released from one reaction to drive the next.
  • The overall $\Delta G$ of a series of reactions is additive.

Factors Influencing Reaction Rate

• Temperature
• Hydrogen ion concentration
• Concentration of reactants

ATP - Energy Carriers

• ATP is used as an energy carrier in the cell
• Energy is stored in the phosphodiester bonds

Components of the Electron Transport Chain (ETC)

• Complex II is not required for oxidative phosphorylation because it does not span the mitochondrial membrane. This complex accepts succinate and is coupled with $FADH_2$ oxidation.
• CoQ is not membrane-bound and can move freely along the membrane
  • Quinol: Fully reduced form contains 2 electrons and 2 protons
  • Quinone: fully oxidized
  • Semiquinone: have reduced forms contains 1 electron
• Cytochrome c is also mobile

Review Oxidation and Reduction

• Electrons are passed down an electrochemical gradient, and molecular oxygen is the final electron acceptor.
• Inhibitors: block oxidation, reducing both ATP generation and oxygen consumption.
• Uncouplers: disrupt the mitochondrial membrane (physically or chemically), reducing ATP production but increasing oxygen consumption (e.g., dinitrophenol and UCP protein).

Location of the ETC

• The ETC is located in the mitochondrial membrane.
• Mitochondrial membrane is impermeable to many compounds.
• $H^+$ cannot pass through the inner mitochondrial membrane, essential for maintaining the $H^+$ gradient needed for ATP production.
• Oxygen is the final electron acceptor.
• NADH or $FADH_2$ generated in the cytosol must be actively transported into the mitochondria.

Shuttle Systems

1) Glycerophosphate Shuttle (Glycerol 3-phosphate shuttle)

• Moves reducing equivalents of NADH from the cytosol to an FAD in the mitochondrion
  • FAD is a required cofactor glycerol-3 phosphate dehydrogenase
  • FAD is tightly or covalently bound to the dehydrogenase.
• Cytosolic dihydroxyacetone phosphate is reduced to glycerol 3-phosphate → glycerol 3-phosphate moves into the mitochondria and transfers the e- to FAD bound to the dehydrogenase

2) Malate-Aspartate Shuttle

• Oxaloacetate DOES not move across the mitochondrial membrane!
• Starting in the cytosol:
  • Oxaloacetate is REDUCED to malate by cytosolic malate dehydrogenase
  • The shuttle moves malate (reduced form of oxaloacetate) into the mitochondria
• Once in the mitochondria:
  • Malate can be OXIDIZED back to oxaloacetate by mitochondrial malate dehydrogenase
• The resulting oxaloacetate can undergo a TRANSAMINATION REACTION with glutamate
  • The reaction transfers the $NH_3+$ from glutamate TO oxaloacetate to generate
    • Both alpha-ketoglutarate and aspartate
  • Aspartate can move in / out of the mitochondria
    • Aspartate can be converted to oxaloacetate.
• For each NADH equivalent → 2.5 molecules of ATP are generated

Enzyme Regulation

• Enzyme quantity
  • Rates of degradation (protein turn over) can impact reaction rates
  • Transcriptional regulation
    • Increase in transcription → increase in enzyme production → increase in the product formed

Allosteric Effectors

• Allosteric compounds bind to a site that is NOT the active site
  • Allosteric activators or positive allosteric effectors enhance an enzyme reaction
    • Stabilize a conformation of the protein that increases binding of substrate and reaction rate. (R-State)
    • Bind to a site that is not the active site an increase enzyme activity
      • Example: AMP can bind phosphofructokinase I and increase its activity
  • Allosteric inhibitors or negative allosteric effectors or modulators inhibit the enzyme reaction
    • Allosteric inhibitors bind to the enzyme at an allosteric site and stabilize a conformation of the protein that decreases binding of substrate and reaction rate. (T state)

Covalent Modifications

• Phosphorylation by a kinase on the R-groups of serine and tyrosine (and sometimes threonine)
  • Phosphorylation can:
    • change the conformation and the activity of a protein, or charge (phosphate groups are negatively charged)
  • Kinases phosphorylate serine/threonine and tyrosine kinases phosphorylate tyrosine residues.
  • Requires: ATP
• Protein phosphatases are enzymes that hydrolyze the phospho-ester bonds of phosphor-seryl and phosphor- tyrosyl residues (R-groups).
  • Dephosphorylation changes the conformation of the protein back to the state it was in before phosphorylation.
  • Dephosphorylation requires: water and $Mg^{2+}$
• Methylation: nucleotide methylation can modify the DNA. Methylation is typical on C or G nucleotides.
• Acetylation: Histone acetylation will alter the condensation of the DNA. Increase histone acetylation will result in a decreased histone:DNA interaction allowing for transcriptional accessibility
• Cleavage
  • Removal of a propeptide or cleavage is often required for activation
  • Ensure that a protein is active in the correct tissue or cellular compartment
  • Examples: chymotrypsinogen to chymotrypsin and proinsulin to insulin

Feedback Inhibition

• Suppression of the activity of an enzyme participating in a sequence of reactions
  • When the product of a reaction accumulates in a cell beyond an optimal amount, its production is decreased by inhibition of an enzyme involved in its synthesis

Feedforward Activation

• The control of a metabolic pathway by a metabolite (of the pathway that acts) in the same direction as the metabolic flux
  • Upstream products can increase the rate of a downstream reaction.

Hemoglobin Conformations

• Hemoglobin can exist in two conformations: T-state or the R-state.
  • In the T (tense) state, Hb has a low affinity for $O_2$.
    • Allosteric inhibitors stabilize the T conformation
  • In the R (relaxed) state, Hb has a high affinity for $O_2$.
    • Allosteric activators stabilize the R conformation
  • Salt bridges are broken between the transitions between the T and R state
• Bohr effect: the impact of pH on oxygen binding hemoglobin. A decrease in pH decreases hemoglobin saturation
  • Effectors to know: 2,3-bisphosphoglycerate (BPG), pH, protons and $CO_2$

Enzymes Kinetics

• The substrate specificity of an enzyme is the ability of an enzyme to select one or a few substrates from a group of similar substrates.
  • The active site contains functional groups that participate in the reaction.
  • The reaction takes place away from water solution.
  • The enzyme usually changes conformation due to the interactions between the amino acid side chain groups of the enzyme and the functional groups of the substrate, so that the outside solution can't take part in the reaction
  • Enzymes increase the rate of the reaction by decreasing the activation energy, i.e., stabilizing the transition state.

General Classes of Enzymes

1. Oxidoreductase: Oxidoreductases catalyze oxidation reduction reactions. At least one substrate becomes oxidized and at least one substrate becomes reduced.
2. Transferase: Transferases catalyze group transfer reactions - the transfer of a functional group from one molecule to another.
   • Example: amino acid transferases use ping-pong reaction that requires pyridoxal phosphate
3. Hydrolase: In hydrolysis reactions, C-O, C-N, and C-S bonds are cleaved by addition of $H_2O$ in the form of $OH^-$ and $H^+$ to the atoms forming the bond.
   • Example: chymotrypsin
4. Lyase: Lyases cleave C-C, C-O, C-N, and C-S bonds by means other than hydrolysis or oxidation.
   • Example: Cleavage of fructose 1,6 bisphosphate by aldolase B to dihydroxyacetone and glyceraldehyde 3-phosphate
5. Isomerase: Isomerases rearrange the existing atoms of a molecule to create isomers of the starting material.
6. Ligase: Ligases synthesize C-C, C-S, C-O, and C-N bonds in reactions coupled to the cleavage of high energy phosphate bonds in ATP or some other nucleotide.

Examples of Clinically Relevant Enzymes as Diagnostics

Cofactors

Three general categories of cofactors are:

• Coenzymes: any organic cofactor that binds to the enzyme and is necessary for the reaction. They are usually inert when not bound to their respective enzyme
• Metal ions: inorganic and may be incorporated as part of a prosthetic group (e.g., heme)
• Prosthetic groups: tightly bound within an enzyme through non-covalent mechanisms

Coenzymes/prosthetic groups to know:

• NAD / FAD: used in redox reactions
• Pantothenic acid (CoA): acyl group carriers
• Thiamine (thiamine pyrophosphate): decarboxylation reactions
• Pyridoxal phosphate: transamination
• Biotin: carboxylation reactions
• Cobalamin: Carbon transfers
• Heme: required for oxygen carrying

Enzymes as Catalysts

• The substrate specificity of an enzyme is the ability of an enzyme to select one or a few substrates from a group of similar substrates.
  • The active site contains functional groups that participate in the reaction.
  • The reaction takes place away from water solution.
  • The enzyme usually changes conformation due to the interactions between the amino acid side chain groups of the enzyme and the functional groups of the substrate, so that the outside solution can't take part in the reaction
  • Enzymes increase the rate of the reaction by decreasing the activation energy, i.e., stabilizing the transition state.

Lock and Key Model

• It was originally thought that a rigid substrate would slide into a rigid active site of the enzyme and a reaction would take place (as a key fits into its matching lock and then works to open the lock).

Induced Fit Model

• The enzyme changes its conformation when it binds to a substrate and this "induced" conformation is due to the interactions between the amino acid side chains of the active site and the functional groups of the substrate; the substrate also changes conformation in response to the enzyme.

Transition State

• State during an enzyme reaction when an intermediate that resembles both substrate and product, and contains the most free energy, exists. The enzyme stabilizes the transition state by lowering its activation energy.

Activation Energy

• Energy necessary to achieve the transition state. Since the rate at which a substrate can become product depends on the rate at which the enzyme/substrate complex can reach the transition state, the activation energy determines how fast a reaction will go.

Reactions types

• Acid- base catalysis activates the substrate by interaction with an acidic or basic amino acid R group to initiate a reaction
  • Specific – reactions are only modified by changes in the concentration of the acid or base participating in the reaction.
  • General – reactions are responsive to all changes in acids or bases
• A covalent catalysis relies on the enzyme becoming modified (typically by a cofactor) and therefore it becomes a reactant in the reaction. The activation energy is reduced and therefore the reaction is faster
  • Example: transamination using pyridoxal phosphate
  • Covalent catalysis is typically facilitated by amino acids that can act as nucleophiles such as: histidine, aspartate, serine and cysteine.
• Catalysis by proximity
  • The higher the concentration of reactants the more likely they will interact.
• Catalysis by strain
  • Cleavage through physical distortion by mimicking the transition state intermediate