Module 10: Biomolecules

Enzyme Characteristics and Mechanisms

Catalytic Nature: Enzymes are biological catalysts composed of proteins that accelerate chemical reactions by lowering activation energy without being consumed, increasing rates by up to $10^{20}$ times under physiological conditions.
Efficiency Example: Carbonic anhydrase catalyzes $\text{CO}_2 + \text{H}_2\text{O} \rightarrow \text{H}_2\text{CO}_3$ to generate $36\text{ million}$ molecules per minute, preventing toxic $\text{CO}_2$ accumulation.
Catalytic Specificity:
- Absolute specificity: Catalyzes only one reaction of a single substance (e.g., urease hydrolyzing the amide bond of urea).
- Relative specificity: Catalyzes reactions of structurally related substances (e.g., proteases hydrolyzing proteins).
- Stereochemical specificity: Catalyzes reactions of only one specific 3D stereoisomer (e.g., D-amino acid oxidase will not react with L-amino acids).
Enzyme Regulation: Cells regulate chemical activity by selectively synthesizing only the specific enzymes needed for their designated function.
Nomenclature:
- Composition-based: End in "-in" (e.g., pepsin, trypsin).
- International Enzyme Commission (IEC): Systematic names describing substrate, functional group, and reaction type across six classes (e.g., urea amidohydrolase).
- Common names: Substrate root plus "-ase" (e.g., urease, alcohol dehydrogenase).
Models of Action:
- Lock-and-key model: The active site is a rigid structure matching the exact complementary shape of the substrate.
- Induced-fit model: The active site is conformationally flexible and adjusts its shape to envelope the incoming substrate.
Enzyme Co-factors:
- Co-factor: A non-protein helper ion or molecule essential for catalytic activity.
- Co-enzyme: An organic co-factor.
- Apoenzyme: The inactive, non-functional protein portion remaining after co-factors or co-enzymes are removed.

Induced-fit model showing enzyme conformational change

Factors Regulating Enzyme Activity and Inhibition

Enzyme Concentration: Direct linear increase in reaction rate with increasing enzyme concentration, assuming excess substrate.
Substrate Concentration: Reaction rate increases until all enzyme active sites become saturated, at which point the rate remains constant.
pH Influence: Enzymes operate maximally around physiological pH ( $\sim 7$ ); deviations lead to denaturation, though specialized enzymes operate in extremes (e.g., stomach enzymes at $\sim \text{pH } 1$ ).
Temperature Influence: Optimum activity occurs around physiological temperature ( $37\,^\circ\text{C}$ ); denaturation occurs at extreme cold ( $0\,^\circ\text{C}$ ) or heat ( $60\,^\circ\text{C}$ and above).
Irreversible Inhibition: Permanent inactivation through covalent binding to essential functional groups:
- $\text{CN}^-$ (cyanide) binds iron in cytochrome oxidase, halting cellular respiration.
- Heavy metal ions ( $\text{Hg}^{2+}$ , $\text{Pb}^{2+}$ ) bind cysteine $-\text{SH}$ groups, precipitating the protein.
Reversible Inhibition: Temporary regulation that prevents excessive catalysis:
- Competitive inhibition: Inhibitor possesses a molecular structure similar to the substrate and competes directly for the active site.
- Non-competitive inhibition: Inhibitor binds an allosteric site away from the active site, inducing a conformational shift that alters the active site shape.

Diagnostic Enzymes in Medicine

Diagnostic Principle: Cell damage or lysis releases tissue-specific intracellular enzymes into bodily fluids above baseline replacement levels, allowing diagnosis via blood serum assays.
Serum Assays and Indications:
- Alanine aminotransferase (ALT): Serum; Hepatitis.
- Acid phosphatase: Serum; Prostate cancer.
- Alkaline phosphatase (ALP): Serum; Liver or bone disease.
- Amylase: Serum; Pancreatic disease.
- Aspartate aminotransferase (AST): Serum, Cerebrospinal fluid; Heart attack or hepatitis.
- Lactate dehydrogenase (LDH): Serum; Heart attack.
- Creatine phosphokinase (CK): Serum; Heart attack.
- Phosphohexose isomerase (PHI): Serum; Heart attack.

Nucleic Acid Components and DNA Structure

Nucleotide Subunits: Monomers comprising a heterocyclic basic amine, a monosaccharide, and a phosphate group.
Nitrogenous Bases:
- Pyrimidines: Single-ring amines consisting of Cytosine (C, both DNA and RNA), Thymine (T, DNA only), and Uracil (U, RNA only).
- Purines: Fused double-ring amines consisting of Adenine (A) and Guanine (G) in both DNA and RNA.
Monosaccharide Components: $\beta$ -configuration pentoses:
- D-deoxyribose in DNA (lacks an oxygen at carbon $2'$ ).
- D-ribose in RNA.
Phosphate Group: Derived from phosphoric acid ( $\text{H}_3\text{PO}_4$ or $\text{HPO}_4^{2-}$ at pH 7); links monosaccharides via phosphate ester bonds, while bases attach via N-glycosidic bonds.
Primary Structure: Unbranched polymer of $1\text{ to }100\text{ million}$ nucleotides linked along an alternating phosphate-monosaccharide backbone; sequences are read from $5'$ to $3'$ .
Secondary Structure: Antiparallel right-handed double helix where the sugar-phosphate backbone faces outward and heterocyclic bases orient inward.
Complementary Base Pairing:
- Adenine pairs with Thymine ( $\text{A}=\text{T}$ ) via two hydrogen bonds ( $\text{O}-\text{H}$ and $\text{H}-\text{N}$ ).
- Guanine pairs with Cytosine ( $\text{G}\equiv\text{C}$ ) via three hydrogen bonds (two $\text{O}-\text{H}$ and one $\text{H}-\text{N}$ ).

Hydrogen bonding between thymine-adenine and cytosine-guanine base pairs

DNA Replication

Semiconservative Mechanism: Each newly formed double helix consists of one original parental strand and one newly synthesized daughter strand.
Step 1 (Unwinding): Helicase unwinds the double helix, creating a replication fork to expose single-stranded templates.
Step 2 (Synthesis): Complementary deoxyribonucleotides are added in the $5' \rightarrow 3'$ direction:
- Leading strand: Synthesized continuously toward the advancing replication fork.
- Lagging strand: Synthesized discontinuously away from the replication fork as Okazaki fragments, which are subsequently joined.
Replication Fidelity and Mutation: Replicative errors occur at an average rate of 1 in $10^{10}$ base pairs; mutations in the genetic code can produce beneficial, neutral, or non-functional protein products.

RNA and Protein Synthesis

RNA Distinctions: Single-stranded nucleic acid containing D-ribose and Uracil (instead of Thymine); located within the nucleus, cytoplasm, and ribosomes.
Classes of RNA:
- Messenger RNA (mRNA): Short-lived transcript that carries genetic information from nuclear DNA to ribosomes.
- Ribosomal RNA (rRNA): Structural and catalytic component making up approximately $65\%$ of ribosomes.
- Transfer RNA (tRNA): Transports specific amino acids from the cytoplasm to the ribosome using complementary anticodons.
Genetic Code: Triplet nucleotide codons ( $4^3 = 64$ permutations):
- Degenerate coding: Multiple codons code for the same amino acid (e.g., Alanine has 4: GCA, GCC, GCG, GCU; Tryptophan has 1: UGG).
- Nonsense (Stop) codons: UAG, UAA, and UGA signal the termination of translation.
Transcription: RNA polymerase opens the DNA at the target gene site; ribonucleotides assemble along the DNA template to synthesize a complementary mRNA strand ( $5' \rightarrow 3'$ ), after which DNA rewinds.
Translation Stages:
- Initiation: Initiation factors recruit mRNA, the ribosomal subunits, and initiator tRNA.
- Elongation: Incoming tRNA enters the aminoacyl (A) site; a peptide bond forms between the amino acid and the growing chain held at the peptidyl (P) site; the empty tRNA is ejected as the ribosome translocates.
- Termination: Encountering a stop codon triggers release factors to sever the completed polypeptide from the tRNA, disassembling the complex.

Elongation phase of translation on the ribosome

Practice Exam Questions and Base Pairing Solutions

Worked Example 1:
- Original sequence ( $5' \rightarrow 3'$ ): CCAT
- Complementary strand ( $3' \rightarrow 5'$ ): GGTA
- Conventional representation ( $5' \rightarrow 3'$ ): ATGG
Worked Example 2:
- Original sequence ( $5' \rightarrow 3'$ ): TGTAATC
- Complementary strand ( $3' \rightarrow 5'$ ): ACATTAG
- Conventional representation ( $5' \rightarrow 3'$ ): GATTACA
Core Concept Review Topics:
- Contrast the active site flexibility of the lock-and-key model with that of the induced-fit model.
- Define the biological distinctions among an apoenzyme, a co-factor, and a co-enzyme.
- Explain the denaturation of enzyme secondary and tertiary structure caused by extreme temperature and pH.
- Differentiate the active site blocking of competitive inhibitors from allosteric distortion by non-competitive inhibitors.
- Contrast primary nucleic acid covalent bonding (phosphodiester and N-glycosidic) with secondary structure stabilization (hydrogen bonding).