Enzymology Notes

Enzymology

Learning Outcomes

• Describe the difference between cofactors and coenzymes.

• Summarize the different roles of vitamins and metals in enzyme function.

• Explain the reactions involving cofactors and coenzymes with enzymes.

Enzymes as Catalysts

• Enzymes dramatically speed up reactions in water at room temperature.

• They catalyze specific bond formations without affecting other functional groups.

• Enzymes are often enantioselective.

• Chemists use enzymes to catalyze reactions due to their efficiency and specificity (Frances Arnold, 2018 Chemistry Nobel prize).

Definition and Classification

• Cofactors/coenzymes are non-protein chemical compounds required for an enzyme's catalytic role.

• They act as helper molecules in biochemical transformations.

• Role:

  • Lower activation energy.

  • Enable reactions to occur efficiently (frequently and faster).

Coenzymes

• Coenzymes are typically small, simple organic non-protein compounds (e.g., pyridoxal phosphate (PLP)).

• They specifically bind to enzyme macromolecules and actively participate in catalytic biotransformations.

• Apoenzymes (protein structures) are inactive without a coenzyme partner; association is synergistic.

• Holoenzyme: Formed when a cofactor or coenzyme binds to the enzyme’s active site, activating the enzyme.

Synergistic Strategy

• Promotes:

  • Site-specific oxidation and reduction.

  • Group transfer reactions (acylation, phosphorylation, methylation).

• These reactions cannot be achieved by enzymes solely based on a protein scaffold.

• Coenzymes played a key role in the creation of complex metabolic networks during the evolution of life.

Holoenzyme vs. Apoenzyme

• Holoenzyme: Enzyme complete with its cofactor or coenzyme.

• Apoenzyme/Apoprotein: Amino acid component of the enzyme without the prosthetic group; inactive.

Holo and Apo Structure

• Human 5-lipoxygenase (5-LOX) catalyzes the oxygenation of polyunsaturated fatty acids.

Amino Acid Biosynthesis

• Aspartate family amino acid biosynthesis requires specific starting building blocks and coenzymes.

• Coenzymes play essential roles in amino acid biosynthesis.

Enzyme Regulation

• Enzymes can be regulated by covalent attachment of chemical groups (e.g., phosphorylation, glycosylation).

• These modifications activate or inactivate the enzyme.

• These chemical modifications are distinct from coenzyme function; they are post-translational modifications.

Glycogen Phosphorylase Regulation

• Glycogen phosphorylase is a homodimer.

• Active form: phosphorylase a, with two specific Ser residues phosphorylated.

• Less active form: phosphorylase b, with unmodified Ser residues.

• Modification is by phosphorylase kinase; demodification (phosphate removal) is by phosphorylase phosphatase.

Cofactors: Metals and Coenzymes

• Cofactors are subdivided into metals and small organic molecules (coenzymes).

• Metal ions are common cofactors.

• Tightly bound cofactors are called prosthetic groups.

Prosthetic Groups

• Tightly integrated into the enzyme structure by covalent or non-covalent forces.

• Organic:

  • Pyridoxal phosphate

  • Flavin mononucleotide (FMN)

  • Flavin adenine dinucleotide (FAD)

  • Thiamin pyrophosphate (TPP)

  • Biotin

• Inorganic:

  • Metals (Co, Cu, Mg, Mn, Zn, Fe)

Role of Metal Ions

• Metalloenzymes: Enzymes containing tightly bound metal ions.

• Metal-activated enzymes: Enzymes requiring loosely bound metal ions as cofactors.

• Metal ions facilitate:

  • Binding and orientation of the substrate.

  • Formation of covalent bonds with reaction intermediates.

  • Interaction with substrate to render them more electrophilic or nucleophilic.

Examples of Metalloenzymes

• Zn++: Carbonic anhydrase, Alcohol dehydrogenase, Carboxypeptidase

• Fe+++ or Fe++: Cytochromes

• Cu++ or Cu+: Cytochrome oxidase

• K+: Propionyl CoA carboxylase

• Mg++: Hexokinase

• Mn++: Superoxide dismutase

• Se: Glutathione peroxidase

• Mo: Xanthine oxidase

• Ni++: Urease

Metal-Activated Enzymes

• Certain ions increase reaction rates in enzyme-controlled reactions.

• Ions may combine with the enzyme or the substrate.

• Ion binding facilitates enzyme-substrate complex formation by affecting charge distribution or the complex's shape.

• Example: Carbonic anhydrase facilitates CO2 transport from tissue to lungs; $HCO_3$ binds to Zinc ion, forming hydrogen bonds to Thr199, leading to weak interaction and fast product dissociation (Kim, J.K., et al., Nat Commun 11, 4557 (2020)).

Amylase Example

• Amylase catalyzes the breakdown of maltose molecules.

• Functions properly only with chloride ions present.

• Without chloride ions, amylase cannot catalyze the reaction:

  • $Starch + Cl^- + \alpha-amylase \rightarrow maltose + intermediate formation of dextrins$

Organic Cofactors (Coenzymes)

• Act as carrier molecules.

• Carbon-based molecules (e.g., FAD, NAD, NADH, riboflavin, plastoquinone).

  • $NADH \rightarrow NAD^+ + H^-$ (carries electron)

  • $Pyruvate + NADH \rightarrow lactic \, acid + NAD^+$ (Lactate dehydrogenase)

Coenzymes as Recyclable Shuttles

• Serve as recyclable shuttles/group transfer agents to transport substrates.

• Water-soluble vitamin B provides essential components of numerous coenzymes.

Chemical Moieties Transported by Coenzymes

• Hydrogen atoms or hydride ions.

• Methyl groups (folates).

• Acyl groups (coenzyme A).

• Oligosaccharides (dolichol).

Classification of Coenzymes: Class-1

• Transport of hydrogen and electrons.

  • Nicotine adenine dinucleotide (NAD+): Electron (hydrogen atom); Lactate dehydrogenase & Niacin derivative

  • Nicotine adenine dinucleotide phosphate (NADP+): Electron (hydrogen atom); Glutamate dehydrogenase & Niacin derivative

  • Flavin adenine dinucleotide (FAD): electron (hydrogen atom); Monoamine oxidase & riboflavin (vit. B2) derivative

Classification of Coenzymes: Class-2

• Transport of groups other than hydrogen and electrons.

  • Coenzyme A (CoA): Acyl groups; Acetyl CoA carboxylase

  • Thiamine pyrophosphate (vit. B1): Aldehydes; Pyruvate dehydrogenase complex

  • Pyridoxal phosphate (vit B6): amino and many other alkyl groups; Transaminases, Decarboxylases, Glycogen phosphorylase

  • Biotin: Carboxyl; Pyruvate carboxylase

  • 5'-Deoxyadenosyl cobalamine (vit. B12): Methylmalonyl mutase

  • Tetrahydrofolate (Folic acid): One carbon compounds; Thymidylate synthase

NAD+ and NADH

• Coenzyme: Organic non-protein molecule, small in size, carries chemical groups between enzymes, acts as an electron carrier.

• NADH (Nicotinamide Adenine Dinucleotide) and FADH2 (Flavin Adenine Dinucleotide) are utilized in almost all biochemical pathways.

• Act as electron carriers; participate in oxidation-reduction reactions.

• NADH: Derivative of Vitamin B3 (Niacin/Nicotinamide); FADH2: Derivative of Vitamin B2 (Riboflavin).

Structures of NAD+ and NADH

• NADH synthesized from Vitamin B3 (Niacin); composed of ribosylnicotinamide 5′- diphosphate coupled to adenosine 5′-phosphate.

• Serves as an electron carrier, converting to oxidized (NAD+) and reduced (NADH) forms.

• Reduced NADH acts as an electron donor, oxidizing to NAD+ while reducing the other compound.

• Involved in glycolysis, TCA cycle, and electron transport chain.

Properties of NADH

• Melting point: 140.0 – 142.0 °C; synthesized in the body, not an essential nutrient.

• Deficiency of Niacin (Vitamin B3) can decrease NADH composition.

• Produced in the cytosol and mitochondria.

• Mitochondrial membrane is impermeable to NADH, distinguishing cytoplasmic and mitochondrial NADH stores.

FAD and FADH2

• FADH2 synthesized from water-soluble vitamin B2 (Riboflavin); reduced form of flavin adenine dinucleotide (FAD).

• FAD synthesized from riboflavin and two ATP molecules; riboflavin phosphorylated by ATP to produce riboflavin 5′- phosphate (flavin mononucleotide, FMN).

• FAD formed from FMN by transfer of an AMP molecule from ATP.

Function of FADH2

• Involved in both carbohydrate metabolism and fatty acid metabolism.

• In carbohydrate metabolism, FADH2 is involved in harvesting high-energy electron-rich fuels in the TCA cycle.

• Generated in each round of fatty acid oxidation; fatty acyl chain shortened by two carbon atoms to yield Acetyl Co A. FADH acts as an electron donor in the electron transport.

Electron Transport Chain

• FADH acts as an electron donor in the electron transport

Similarities Between NADH and FADH2

• Both are coenzymes.

• Both act as electron carriers.

• Both are nonprotein organic molecules.

• Both are derived from vitamins.

• Both are water-soluble.

• Both can exist in reduced and oxidized forms.

• Both participate in oxidation and reduction reactions, transferring electrons.

• Both can be synthesized in the body.

• Both take part in metabolic pathways (carbohydrate, fatty acid, amino acid, nucleotide metabolism).

Example of FAD-dependent enzyme

• Carbohydrate metabolism

  • Pyruvate dehydrogenase complex: Pyruvate to Acetyl CoA

  • α-Ketoglutarate dehydrogenase complex: α-Ketoglutarate to Succinyl CoA

  • Succinate dehydrogenase: Succinate to Fumarate

• Lipid metabolism

  • Acyl CoA dehydrogenase: Acyl CoA to α, β-Unsaturated acyl CoA

• Protein metabolism

  • Glycine oxidase: Glycine to Glyoxylate + $NH_3$

  • D-Amino acid oxidase: D-Amino acid to α-Keto acid + $NH_3$

• Purine metabolism

  • Xanthine oxidase: Xanthine to Uric acid

Role of Vitamins

• Vitamins are crucial in various metabolic pathways, including:

  • Glycogenolysis (B6)

  • Fatty Acid Degradation (B2, B3, B5)

  • Gluconeogenesis (B3)

  • Glucose-6-P (B3, B5)

  • Pentose Pathway (B1)

  • Glycolysis (B1, B2, B3, B5)

  • Fatty acid synthesis (B7)

  • Amino Acid Degradation (B3, B6)

  • One Carbon Metabolism (B2, B9, B12)

  • Electron Transport Chain (B2, B3)

Functions of Riboflavin (Vitamin B2)

• Involved in energy production.

• Helps keep mucus membranes in the GI tract moist.

• Keeps skin, hair, eyes, and nervous system in good condition.

• Helps repair damaged tissue and wounds.

• Involved in processing amino acids and fats.

• Active in the formation of red blood cells.

• May help prevent migraines.

• Riboflavin helps our body to covert nutrients into energy and it is important role in therapeutic approaches for various inborn errors of metabolism. Riboflavin supplementation improve disorders related to riboflavin metabolism (Brown-Vialetto-Van Laere Syndrome, Fazio-Londe syndrome) and disorders related with flavin dependent enzymes (SCADD, MCADD, GA-1, RR-MADD).

Photosynthesis

• Role of Plastoquinone

Coenzyme A (CoA)

• CoA carries acyl groups; common in metabolic reactions.

• Reacts with carboxylic acids to form thioesters, functioning as an acyl group carrier.

• Assists in transferring fatty acids from the cytoplasm to mitochondria.

• Acyl-CoA: Coenzyme A molecule carrying an acyl group.

Pyruvate Conversion

• Pyruvate is converted into acetyl-CoA before entering the citric acid cycle.

Inorganic Cofactors

• Involved in direct catalysis; stabilize the enzyme.

• Do not strictly carry molecules like coenzymes.

• Help the enzyme to convert one substrate to another.

• Mostly metals.

• Example: Mg2+ as a cofactor to stabilize negative charge on DNA polymerase.

Inorganic Cofactors (Examples)

• $Fe^{2+} \, or \, Fe^{3+}$: Cytochrome oxidase, Catalase, Peroxidase

• $Cu^{2+}$: Cytochrome oxidase

• $Zn^{2+}$: DNA polymerase, Carbonic anhydrase, Alcohol dehydrogenase

• $Mg^{2+}$: Hexokinase, Glucose-6-phosphatase

• $Mn^{2+}$: Arginase

• $K^{+}$: Pyruvate kinase (also requires Mg2+)

• $Ni^{2+}$: Urease

• Mo: Nitrate reductase

• Se: Glutathione peroxidase

Four Common Features of Enzymes

• Reaction occurs much faster.

• Enzyme molecule is not permanently altered by the reaction.

• Can catalyze both the forward and the reverse reaction.

• Highly specific for the substrates they bind.

Detailed Explanation of Enzyme Features

• Enzymes do not initiate reactions that would not occur on their own; they only accelerate them.

• The enzyme molecule is not permanently altered and can be reused repeatedly.

• Enzymes catalyze both forward and reverse reactions, although one direction may be more favorable.

• Enzymes exhibit high specificity for substrates, catalyzing only one reaction.

Enzyme Activity

• The presence of coenzymes or inhibitors will influence the activity of the enzymes.

Summary

• Not all enzymes function alone; they often need help.

• Coenzymes act as carrier molecules.

• Cofactors assist with catalysis.

• Vitamins and minerals are dietary cofactors and coenzymes.

Coenzymes, Cofactors, and Prosthetic Groups

• Coenzymes: Reusable organic non-protein molecules that carry carbon and bind loosely to catalyze reactions.

• Cofactors: Reusable inorganic non-protein molecules (metal ions) that loosely bind and must be supplemented in the diet.

• Prosthetic groups: Organic vitamins, sugars, lipids, or inorganic metal ions that bind tightly or covalently to enzymes to aid in catalysis; used in cellular respiration and photosynthesis.