Cellular Energy and Enzymes

Cell Requirements

• Cells need a way to encode/transmit information
• They also require a membrane that separates the inside from the outside
• Energy is a necessity for cells

Adenosine Triphosphate (ATP)

• ATP stores energy that cells readily use to perform work
• Hydrolysis of ATP drives many cellular reactions

Gibbs Free Energy (ΔG)

• Gibbs free energy is the amount of energy available in a system for work
• ΔG is the difference in energy between reactants and products in a chemical reaction
• If Products > Reactants, ΔG is positive
• If Products < Reactants, ΔG is negative
• Endergonic reactions have a positive ΔG and absorb energy
• Exergonic reactions have a negative ΔG and release energy
• ATP hydrolysis is an exergonic reaction that releases energy, with ΔG = -7.3 kcal/mol

Enzymes

• Enzymes decrease activation energy, speeding up reactions
• They are 3D structures with particular amino acids forming an active site
• The active site binds to a substrate, converting it into a product

Enzyme Characteristics

• Enzymes enable chemical reactions at rates needed for cell survival
• Enzymes are generally proteins that can be regulated
• Enzyme activity relies on inhibitors that turn off enzymes and activators

Competitive vs. Allosteric Inhibitors

• Competitive inhibitors bind to the enzyme's active site, competing with the substrate
• Allosteric inhibitors bind to a site other than the active site and change the enzyme's shape

DNA Replication

• DNA replication is semiconservative, producing a new strand using a single parent strand
• DNA replication involves a leading strand and a lagging strand

Leading vs. Lagging Strand

• Leading Strand: 3' end points toward the replication end and is synthesized as one long, continuous strand
• Lagging Strand: 3' end points away from the replication end and is synthesized in short, discontinuous pieces

Enzymes in DNA Replication

• Helicase unwinds the parental DNA double helix at the replication fork
• Single-stranded binding proteins prevent single-stranded regions of DNA from re-annealing
• Topoisomerase relieves stress from unwinding the DNA double helix
• RNA primase synthesizes a short RNA piece complementary to the DNA template, enabling DNA polymerase to start
• DNA polymerase synthesizes DNA
• Ligase joins discontinuous DNA fragments

Point Mutations

• Point mutations are changes in a single nucleotide

Nonsynonymous vs. Nonsense Mutations

• Nonsynonymous mutations: an amino acid changes due to mutation, and the protein misfolds and no longer functions
• Nonsense mutations: a codon changes to a STOP codon, resulting in a non-functional polypeptide

Frameshift Mutations

• Frameshift mutations involve insertions or deletions that are not multiples of three, disrupting the reading frame

Cell Cycle Phases

• G1 Phase: Size and protein content (e.g., DNA polymerase) increases to prepare for S phase
• S Phase: DNA is replicated
• G2 Phase: Cell prepares for mitosis (M phase)

Regulation of the Cell Cycle

• Cyclin D-CDK prepares the cell for S phase as part of the G1/S Cyclin-CDK complex
• Cyclin A-CDK initiates DNA synthesis and prevents DNA replication more than once per cycle, as part of the S Cyclin-CDK complex
• Cyclin B-CDK prepares the cell for mitosis as part of the M Cyclin-CDK complex

Cell Cycle Checkpoints

• DNA damage checkpoint checks for damaged DNA before S phase
• DNA replication checkpoint checks for the presence of unreplicated DNA at the end of G2, before mitosis
• Spindle assembly checkpoint checks to make sure all chromosomes attach to the spindle before the cell progresses with mitosis

p53

• p53 is a tumor suppressor gene that encodes a protein and inhibits the cell cycle and cell division
• When DNA is damaged, a protein kinase activates and phosphorylates p53
• In normal conditions, p53 is exported from the nucleus at low levels
• When phosphorylated, p53 is prevented from being exported with high levels in the nucleus
• Increased p53 activates the transcription of a gene that expresses CDK protein
• Arriving at the G1/S transition and gives cells time to repair DNA damage, the inhibitor binds to and blocks activity of G1/S cyclin CDK complex

Polymerase Chain Reaction (PCR) & DNA Replication

• PCR amplifies specific DNA sequences
• Denaturing: Template DNA is heated to separate it into two strands
• Annealing: After cooling, primers bind to complementary sequences on the DNA strands
• Extension: DNA polymerase synthesizes new strands from primers in the 5' to 3' direction. Taq polymerase is a DNA polymerase that works at high temperatures

Recombinant DNA

• Restriction enzymes, which cut DNA at specific sequences, are heavily relied on
• Requires a fragment of double-stranded DNA to act as a donor
• Reverse transcriptase is used to make double-stranded complementary DNA (cDNA) from mRNA for protein-coding genes
• It also requires a vector sequence with the ability to be maintained in the new cell to insert a donor fragment
• DNA is cut with the same restriction enzyme so that both the donor DNA and the vector have complementary ends
• Ligase joins the vector and donor together
• The recombinant DNA is transformed into bacteria and replicated