Enzyme Kinetics and Nucleic Acid Structure Comprehensive Study Guide

Enzyme Kinetics and Nilectrofluidase Experiment

• The lecture begins with a review of a scientific experiment performed on the enzyme Nilectrofluidase.
• The primary tool for analyzing this experiment is the Lineweaver-Burk equation:   $\frac{1}{v} = \frac{K_m}{V_{max}} \times \frac{1}{[s]} + \frac{1}{V_{max}}$
• In this linear equation, $\frac{1}{v}$ corresponds to the y-axis, and $\frac{1}{[s]}$ corresponds to the x-axis ($y = mx + c$).

Step-by-Step Calculation Representative of the Data

• First, calculate the values for $\frac{1}{s}$ and $\frac{1}{v}$.
• Provided data points:
  • $x_1 (1/s_1) = 0.1$
  • $x_2 (1/s_2) = 0.05$
  • $y_1 (1/v_1) = 12$
  • $y_2 (1/v_2) = 11$
• Calculate the slope ($m$):   $m = \frac{12 - 11}{0.1 - 0.05} = \frac{1}{0.05} = 20$
• Calculate the y-intercept ($c$) using $y = mx + c$:   $12 = 20 \times 0.1 + c$$12 = 2 + c$$c = 10$
• Determining Kinetic Parameters:
  • $V_{max}$ is the reciprocal of the y-intercept: $V_{max} = \frac{1}{c} = \frac{1}{10} = 0.1$
  • $K_m$ can be found via the slope: $m = \frac{K_m}{V_{max}} \Rightarrow 20 = \frac{K_m}{0.1} \Rightarrow K_m = 2$
  • Alternatively, $K_m = \frac{m}{c} = \frac{20}{10} = 2$

Example of Inhibition

• In a provided scenario where an inhibitor is present:
  • Initial values: $K_m = 2$, $V_{max} = 0.1$
  • Inhibited values: $K_m = 2$ (remains the same), $V_{max}$ decreases to $0.02$
  • Conclusion: This describes noncompetitive inhibition because the $K_m$ is unchanged while $V_{max}$ decreases.
• The speaker notes that in exams, numerical values will typically be rounded; results such as $11.27$ may indicate a calculation error.

Nucleic Acids and the Central Dogma

• Nucleic acids are essential because they carry genetic material and participate in the central dogma of molecular biology.
• The Central Dogma of Molecular Biology describes the transfer of information:
  • DNA replicates to make another copy of itself.
  • DNA serves as a template for synthesizing mRNA (Transcription).
  • mRNA serves as a template for synthesizing proteins (Translation).
• Historical Context:
  • Watson and Crick elucidated the structure of DNA.
  • Much of the crucial data, specifically the X-ray crystallographic data of DNA fibers, was acquired by Rosalind Franklin.
  • Watson and Crick observed Franklin's data (with the help of Maurice Wilkins) to determine the double-hemical structure.
  • A relevant BBC movie about this discovery is titled "Life Story" (or "Race for the Double Helix").

Nucleotide Structure and Nomenclature

• The basic building block of nucleic acid polymers (DNA and RNA) is the nucleotide.
• A nucleotide consists of three groups:
  1. A nitrogenous base.
  2. A pentose sugar.
  3. A phosphate group.
• Glycosidic Bond: The bond connecting the sugar and the nitrogenous base.
• Nucleoside: A molecule consisting of only the sugar and the base.
• Nucleotide Designation:
  • A nucleoside becomes a nucleotide when connected to a phosphate (e.g., nucleoside monophosphate).
  • Nucleotides can be monophosphates, diphosphates, or triphosphates.

Nitrogenous Bases: Purines and Pyrimidines

• Purines:
  • Structure: Fused five- and six-membered aromatic rings (indole-like ring).
  • Shorter name, larger molecule.
  • Types: Adenine (A) and Guanine (G).
• Pyrimidines:
  • Structure: A single six-membered aromatic ring.
  • Longer name, smaller molecule.
  • Types: Cytosine (C), Thymine (T) (found in DNA), and Uracil (U) (found in RNA).
• Other naturally occurring bases include: Xanthine, Hypoxanthine, and Uric acid (the excretory substance for birds and reptiles).

Numbering Systems

• Purine numbering starts at the top left nitrogen of the six-membered ring and goes counter-clockwise ($1-6$), then proceeds to the five-membered ring ($7-9$).
• Pyrimidine numbering proceeds around the single ring.
• Sugar numbering: Atoms are designated with a "prime" (e.g., $1', 2', 3', 4', 5'$) to distinguish them from the base numbering. The glycosidic bond connects to the $1'$ position.

Properties of Nitrogenous Bases

• Keto-Enol Tautomerization:
  • Bases exist in equilibrium between keto and enol forms.
  • The keto form is the most predominant and stable form ($>95\%$).
• Acid-Base Properties:
  • Cytosine and Adenine can grab a proton (act as bases). Cytosine has a $pKa \approx 4.5$, and Adenine has a $pKa \approx 4.2$.
  • Uracil, Thymine, and Guanine can release protons. Their $pKa$ values are approximately $9.4$ to $9.5$.
• Aromaticity and UV Absorption:
  • All five bases are aromatic and absorb electromagnetic radiation.
  • $\lambda_{max}$ for nucleic acids is $260\,nm$.
  • This contrasts with proteins, which typically absorb at $280\,nm$.
  • Pure nucleic acid extractions are checked using the $280:260$ ratio; a low ratio indicates protein contamination.
• Solubility:
  • Nitrogenous bases by themselves have very low water solubility because they are aromatic.
  • When conjugated to a sugar (nucleosides), the many hydroxyl (OH) groups on the sugar increase water solubility significantly via hydrogen bonding.

Sugars and Phosphates in Nucleic Acids

• Pentose Sugars:
  1. Ribose: Found in RNA; contains a $2' \text{-OH}$ group.
  2. Deoxyribose: Found in DNA; the $2' \text{-OH}$ group is replaced by a hydrogen (hence "deoxy").
• Nomenclature Changes:
  • Base: Adenine → Nucleoside: Adenosine.
  • Base: Guanine → Nucleoside: Guanosine.
  • Base: Cytosine → Nucleoside: Cytidine.
  • Base: Uracil → Nucleoside: Uridine.
• Nucleotide Types:
  • Phosphate groups can be at the $5'$ position (most common), $3'$ position, or exist as cyclic phosphates (e.g., $5' \text{ to } 3'$ or $3' \text{ to } 2'$).
  • ATP (Adenosine Triphosphate): Central to energy metabolism. Phosphates are labeled alpha ($\alpha$), beta ($\beta$), and gamma ($\gamma$) beginning from the sugar.
  • GTP: Drives protein synthesis.
  • CTP: Drives lipid synthesis.
  • UTP: Drives carbohydrate metabolism.
• Phosphates are polyprotic acids and carry a negative charge at neutral pH.

Polymerization and Chain Structure

• Nucleic acids are linear polymers of nucleotides.
• Linkage: The $3' \text{-OH}$ of one sugar reacts with the $5' \text{-phosphate}$ of the incoming nucleotide to form a phosphodiester bridge.
• Directionality:
  • One end has a free $5' \text{-phosphate}$ (the $5'$ end).
  • The other end has a free $3' \text{-OH}$ group (the $3'$ end).
  • DNA and RNA sequences are always read and written in the $5' \rightarrow 3'$ direction.

RNA Varieties and Functions

• RNA is typically single-stranded but can form complex sub-structures.
• Types include: mRNA (messenger), tRNA (transfer), rRNA (ribosomal), snRNA, siRNA, sRNA, microRNA, and long non-coding RNA.
• Ribosomes:
  • Bacterial ribosome is the 70S (S = Svedberg unit, a sedimentation coefficient).
  • Composed of a large subunit (50S) and a small subunit (30S).
  • The 30S subunit contains 16S rRNA ($\approx 1,500$ nucleotides) and 21 proteins.
• Modified Nucleotides: There are over 100 (approximately 170 known) modified nucleotides in RNA, such as methylations or acylations.

Chemical Stability: DNA vs. RNA

Why Thymine in DNA instead of Uracil?

• Cytosine naturally undergoes deamination, a process where the amine group is replaced by a carbonyl group, turning Cytosine into Uracil.
• If DNA naturally used Uracil, repair enzymes would be unable to distinguish between a "correct" Uracil and a Uracil resulting from Cytosine damage.
• Using Thymine (which is methylated Uracil) allows the cell to identify any Uracil in DNA as a mutation/damage that needs to be repaired to prevent genetic errors.

Why Deoxyribose in DNA?

• The $2' \text{-OH}$ group in Ribose (RNA) acts as a nucleophile. Under basic conditions, it can attack the phosphodiester backbone, leading to self-catalyzed strand cleavage and degradation.
• DNA lacks this $2' \text{-OH}$ group, making the molecule significantly more stable and suitable for long-term genetic storage.
• In RNA, the $2' \text{-OH}$ is necessary for its role as a ribozyme (catalytic RNA).

DNA Conformations and Geometry

• Chargaff's Rules: In DNA, the ratio of A:T is $1:1$ and the ratio of G:C is $1:1$.
• Base Pairing:
  • Watson-Crick pairs: $A-T$ and $G-C$.
  • The distance between sugars in an $A-T$ pair is roughly $1.11\,nm$; for $G-C$, it is roughly $1.08\,nm$.
  • Two strands are antiparallel ($5' \rightarrow 3'$ vs. $3' \rightarrow 5'$).
• Torsional Angles: There are 12 dihedral angles defining the structure, including 6 in the backbone, 5 in the sugar, and the $\chi$ (chi) angle for the glycosidic bond.

Sugar Pucker (Pseudo-rotation Angle)

• Five-membered rings are not flat; they adopt non-planar conformations to minimize strain.
• Envelope form: 4 atoms in plane, 1 atom out.
• Twisted/Crystal form: 3 atoms in plane, 2 atoms out (one up/endo, one down/exo).
• C2'-endo: Two-prime carbon is up. Favorable for B-DNA (standard DNA).
• C3'-endo: Three-prime carbon is up. Favorable for A-DNA and RNA.

Glycosidic Bond Confirmation (Chi Angle)

• Syn-confirmation: The base is oriented over the sugar ring.
• Anti-confirmation: The base is oriented away from the sugar ring.
• Watson-Crick base pairing requires the anti-confirmation.
• Many antiviral drugs (like AZT, the first anti-HIV drug) are nucleotide analogs that prefer the syn-confirmation, which blocks viral replication by causing polymerase abortive transcription.

Helical Forms and Grooves

• B-form DNA: Standard biological DNA, right-handed helix, $10$ base pairs per turn, $C2' \text{-endo}$ sugar pucker.
• A-form DNA/RNA: Right-handed helix, $11$ base pairs per turn, $C3' \text{-endo}$ pucker. Observed in RNA double strands and low-humidity DNA.
• Z-form DNA: Left-handed helix, observed in very high salt or specific laboratory conditions; not naturally common.
• Grooves:
  • Major Groove: Wide and deep. It can accommodate protein structures like an alpha-helix for sequence recognition.
  • Minor Groove: Narrow and shallow.
• Base Pair Deviations: Base pairs are not perfectly flat; they exhibit movements described as tilt, roll, and propeller twist.

Questions & Discussion

Question regarding the role of the 2'-OH group:

• Student: What role does the OH group play if the attachment is through the 3'?
• Response: The 2'-OH makes RNA susceptible to degradation. In DNA, replacing it with hydrogen prevents this degradation, ensuring genetic stability. However, in RNA, this group is vital for its catalytic activity as a ribozyme.

Question regarding exams and reviews:

• Student: Is there a bonus quiz on Friday?
• Response: The instructor will consider a bonus quiz if requested. The second exam is on Monday, with reviews likely on Friday at 8:30 AM or 4:00 PM. Topic 9 will be pushed to the end of the semester as it is "dry."