Introduction to Biochemistry: Enzyme Catalysis and Coenzymes

Introduction to Biochemistry

Course: BCH2333B.
Session: W10A.
Inspirational Quote from Jane Goodall: "What you do makes a difference and you have to decide what kind of difference you want to make."

Enzyme Catalysis and Plastic Degradation

Environmental Context of Plastics:
- Human-made polymers have only been around since the 1950s. Consequently, many microbes have not yet evolved the necessary mechanisms to degrade them.
- Global Production and Waste: Every year, approximately 300 million tonnes of plastics such as Polyethylene (PE), Polypropylene (PP), and Polyethylene terephthalate (PET) are discarded.
- Waste Management Statistics:
  - Only $10\%$ is recycled.
  - $60\%$ is burned or ends up in landfills.
  - $30\%$ pollutes ecosystems, where it can persist for centuries.
Polyethylene Terephthalate (PET) Degradation:
- In 2016, researchers isolated and engineered PET-degrading enzymes from the bacterium Ideonella sakaiensis 201-F6.
- The PET Degradation Process:
  - Step 1: The PETase enzyme degrades the polyester plastic polymer (PET) into monohydroxyethyl terephthalate (MHET).
  - Step 2: The MHETase enzyme degrades MHET into its two primary constituents: ethylene glycol (EG) and terephthalic acid (TPA).
Engineering and Structure:
- PDB ID 7vve: This structure represents an engineered PET-breaking enzyme designed for enhanced plastic-digesting ability and greater thermal stability.
- The structure shows MHET bound in the active site and highlights specific active site residues: $ile$ , $asp$ , $his$ , $ala$ , $gly$ , and $cys$ .
- Mutations in these areas enhance enzyme activity.
- Thermal Challenges: While PET catalysts are being developed, PE and PP only begin to break down above $130^{\circ}C$ . These are temperatures that current enzymes cannot handle.

Five General Mechanisms of Catalysis

Following substrate binding, enzymes utilize five general mechanisms to catalyze product formation. Multiple mechanisms can be employed simultaneously during the conversion of Substrate ( $S$ ) to Product ( $P$ ):

Binding Change Catalysis: A special case includes "induced fit," where the substrate and/or the enzyme undergo structural changes upon binding.
Metal Ion Catalysis: Metal ions are specifically coordinated in the active site to drive reactions through conformational or chemical mechanisms (e.g., acting as a reactive nucleophile).
Catalysis by Approximation: Enhancement of reaction rates occurs by bringing multiple substrates into close proximity and the correct orientation (the proximity effect).
Covalent Catalysis: An active site residue forms a covalent bond with the substrate during the catalytic cycle.
Acid-Base Catalysis: A molecule other than water serves as the proton donor or acceptor.

Binding Change Catalysis: Hexokinase

Enzyme Function: Hexokinase catalyzes the phosphorylation of the 6th carbon of $\beta\text{-D-glucose}$ or $\alpha\text{-D-glucose}$ .
Substrate Specificity: While other sugars like mannose, fructose, and arabinose can act as substrates, glucose is preferentially phosphorylated.
Mechanism of Action:
- Open Configuration: In the absence of substrate, the enzyme is inactive, and no phosphate transfer occurs.
- Closed Configuration: The binding of glucose induces a "closure" of the enzyme. This closed conformation aligns the catalytic residues required for phosphate transfer (kinase activity).
Energetics:
- The binding energy ( $\Delta G_B$ ) results from the stabilization of the transition state ( $TS^{\ddagger}$ ).
- This involve an increase in non-covalent bonding and a decrease in enthalpy ( $\Delta H$ ).
- This energy effectively "pays" for the conformational change required upon substrate binding.

Metal Ion Catalysis: Enolase

Enzyme Function: Enolase catalyzes the reversible dehydration of 2-phosphoglycerate into phosphoenolpyruvate, which are key intermediates in the glycolysis pathway.
Role of Magnesium ( $Mg^{2+}$ ):
- The reaction utilizes two coordinated $Mg^{2+}$ ions to affect the acidity of the $\alpha\text{-C}$ proton.
- The substrate carboxyl group interacts strongly with the active site $Mg^{2+}$ , pulling electron density away from the $\text{C}_{\alpha}$ . This acidifies the $\alpha\text{-C}$ proton, facilitating its abstraction by an attacking Lysine ( $Lys$ ) residue.
- The resulting enolate intermediate is stabilized by the $Mg-CO$ interaction, which shields the two negative oxygen atoms from each other.

Catalysis by Approximation: Glycine N-methyltransferase (GNMT)

Enzyme Function: GNMT catalyzes the methylation of the amine group of glycine to form sarcosine (found in muscle tissues).
Mechanism:
- The reaction involves a methyl donor: S-adenosyl methionine (SAM).
- Precise positioning is essential: the Glycine amine must be placed next to the methyl donor.
- GNMT undergoes a conformational change upon binding Glycine and SAM to bring the reactants into close proximity, facilitating the N-methylation reaction.

Covalent Catalysis: Serine Proteases

Problem: Peptide bond hydrolysis is thermodynamically favorable but extremely slow at biological timescales (taking $10$ to $1000$ years).
Resonance and Stability: The peptide bond possesses a $\pi\text{-like}$ nature (resonance), which limits bond rotation and reduces the nucleophilic susceptibility of the carbonyl carbon.
Protease Mechanism (Step-by-Step):
- Step 1: A strong nucleophile ( $Ser-OH$ ) in the active site performs a nucleophilic attack on the carbonyl carbon of the target peptide bond.
- Step 2: Hydrolysis is completed to release the products.
The Catalytic Triad (Chymotrypsin Example):
- The triad consists of Aspartate 102 ( $D102$ ), Histidine 57 ( $H57$ ), and Serine 195 ( $S195$ ).
- Formation of the Nucleophile:
  1. The carboxyl group of $D102$ Hydrogen-bonds to the $\delta\text{-H}$ of $H57$ .
  2. Upon tautomerization, this results in an electron-deficient, cationic imidazole ring on $H57$ .
  3. The cationic $\epsilon\text{-N}$ of $H57$ creates a dipole-cation interaction that effectively deprotonates $S195$ .
  4. This forms a highly nucleophilic alkoxide ion.
- Specificity Pockets:
  - Active sites are adjacent to a specificity pocket (S1).
  - The S1 pocket recognizes the side chain on the N-terminal of the scissile bond (the bond to be cleaved).
  - Chymotrypsin: Prefers bulky hydrophobic residues ( $F$ , $Y$ , $W$ , $M$ ).
  - Trypsin: Prefers positively charged residues ( $K$ , $R$ ).
  - Elastase: Prefers small residues ( $A$ , $S$ ).

Acid-Base Catalysis and Dopamine

General Principle: Proton transfer is catalyzed by a molecule other than water. This distributes partial charges over a larger network of atoms in the transition state.
Example: Keto-enol Tautomerism: The initiating step is the acid or base-catalyzed transfer of the $\alpha\text{-proton}$ .
Dopamine Biosynthesis Pathways:
- Classical Biosynthesis:
  1. $L\text{-Phenylalanine}$ ( $Phe$ ) $\rightarrow$ $L\text{-Tyrosine}$ ( $Tyr$ ).
  2. $L\text{-Tyrosine}$ is converted to $L\text{-Dihydroxyphenylalanine}$ ( $L\text{-DOPA}$ ) by Tyrosine hydroxylase.
  3. $L\text{-DOPA}$ is converted to Dopamine by DOPA decarboxylase (also known as Aromatic L-amino acid decarboxylase).
- Alternative Biosynthesis: Includes Tyramine-mediated pathways.
- Degradation: Dopamine can be degraded into $3,4\text{-Dihydroxyphenylacetic acid}$ ( $DOPAC$ ) or Homovanillic acid ( $HVA$ ).
Dopamine Pathways in the Brain:
- Mesocortical: Related to cognition, memory, attention, emotional behavior, and learning.
- Nigrostriatal: Related to movement and sensory stimuli.
- Mesolimbic: Related to pleasure, reward-seeking behaviors, addiction, emotion, and perception.
- Tuberoinfundibular: Related to immunity, metabolism, and sexual gratification.
Reward Prediction Error:
- Unexpected Reward: Increases dopamine activity (positive feedback).
- Expected Reward: Dopamine activity shifts to the "cue" rather than the reward itself; the reward has no additional effect.
- Negative Prediction Error: If an expected reward is not received, dopamine activity drops below baseline, weakening the association.

Enzyme Cofactors and Coenzymes

Definitions:
- Cofactor: Usually metals or trace elements that provide properties to the enzyme (metalloenzyme) that amino acid side chains lack. They can be part of prosthetic groups (e.g., Iron in heme) or coordinated directly (e.g., Magnesium in enolase).
- Coenzyme: Complex organic molecules that act as transient carriers of specific functional groups. They are not irreversibly changed (they are either unmodified or regenerated).
Roles of Cofactors:
1. Substrate Alignment and Charge Withdrawal: Metals like $Zn^{2+}$ and $Mg^{2+}$ limit conformational freedom and reduce the degree of charge on anions, lowering $\Delta G^{\ddagger}$ .
2. Oxidation-Reduction: Superoxide dismutase (SOD) uses cofactors like $Cu-Zn$ to neutralize toxic oxygen radicals (superoxide, $O_2 \cdot^-$ ).
Coenzymes and Vitamins:
- Most coenzymes are derived from water-soluble Vitamin-B complex.
- Nicotinamide Adenine Dinucleotide ( $NAD^+$ ): A niacin derivative (Vitamin B3) involved in hydride ion ( $H^-$ ) transfer. It is reduced to $NADH$ . Alcohol dehydrogenase uses $NAD^+$ to convert ethanol to acetaldehyde.
- Pyridoxal Phosphate ( $PLP$ ): Derived from Vitamin B6; essential for DOPA decarboxylase.
- Synthesis of Neurotransmitters: Requires multiple cofactors/coenzymes including Iron, Folate, $BH_4$ , Vitamin D, Vitamin C, Magnesium, Copper, and $SAMe$ .

The Placebo Effect

The placebo effect is well-documented but has historically been viewed negatively or merely as a clinical control.
A survey of US doctors revealed that approximately $50\%$ admitted to prescribing placebos. Similar findings have been confirmed in Canada.
Open-Label Placebos: Recent research suggests that placebos can be effective even when patients are fully aware they are taking a placebo.