Cell Biology Term 2

Enzyme classification

isomerase: rearrangement of atoms

ligase: joining of nucleic acid molecules

protease: digestion of other proteins

Gibbs free energy

When delta G<0 — SPONTANEOUS/ EXERGONIC

When delta G>0 — NON SPONTANEOUS/ ENDERGONIC

When delta G=0 — EQUILIBRIUM

Spontaneous reaction

• exergonic

• releases energy as a product

• positive change in enthalpy

Non spontaneous reaction

• endergonic

• uses energy as a reactant

• negative change in enthalpy

Delta G&S and Biological Systems

• as energy is added, disorder decreases. therefore delta S decreases and delta H increases

Activation Energy

minimum amount of energy required in order for the reactants to react and give rise to a final product

Metastable State

• within a cell, rate of uncatalyzed reactions is low, molecules appear stable (thermodynamically favourable)

• in a metastable state

Transition State

• intermediate stage where free energy is greater than that of initial reactants

• unstable

Must overcome Activation Energy

• metastable state

• transition state

• stable state

  • high activation + metastable state = cellular rxns only occur when appropriate catalyst is present

Catalysts

• provide a location and alignment to help facilitate a reaction

• reduce energy required for reaction

Important properties of catalysts

1. speed up reactions by lowering EN

2. speed up only exergonic reactions

   1. not consumed or changed by reactions
      APPLIES TO ENZYMES TOO

Enzymes as catalysts

• organic catalysis increase reaction speed 10^7-10^17 times

• increase energy content by:

  • input of heat

  • lower EN

Enzymes

• biological catalysts

• have specific binding pockets

• lower EN

Enzymes as Biological Catalysts

• proteins that increase rate of rxn 

• catalyze all chemical runs in body

• have unique 3D shapes that fit shapes of reactants

• catalyze over 4000 runs

Enzymes are Reusable

• enzymes can be reused but eventually degrade

• they are susceptible to change in temp and pH

Active Site of Enzyme

• fits shape of substrate molecules

• a.a side chains align to bind substrate through H bonds, salt bridges, hydrophobic interactions (TRANSIENT BONDS)

• products are released when rxn is complete

Enzyme Specificity

• have a varying degree of specifity

• may recognize and catalyze:

  • a single substrate

  • group of similar substrates

  • particular type of bond

Lock and Key Model

• active sitr has a rigid shape

• only substrates with matching shape can fit

• substrate is a key that fits the lock on active site

Induced Fit Model

• active site is flexible not rigid

• shape of enzyme, active site and substrate adjust to maximize fit

• greater range of substrate specificity

Enzyme Catalyzed Reactions

when a substrate fits in active site: enzyme-substrate complex is formed

within complex, reaction occurs to convert substrate to product

products then released

E + S = ES = E + P

Temperature & Enzyme Activity

• enzymes are most active at optimal temp (37 degrees)

• show little activity at low temps

• activity is lost at high temps as denaturation occurs

pH & Enzyme Activity

• enzymes are most active at optimal pH

• activity is lost at low or high pH as tertiary structure is disrupted

Optimum pH for selected enzymes

• optimal = 7.4

• certain organs cause enzymes to operate at lower or higher pH

Enzyme Kinetics

• how fast substrate is being converted to product

• rate can be increased by increasing substrate or enzyme [ ] 

Enzyme [ ] and Reaction Rate

• rate increases as [ ] increases (with constant substance [ ])

• linear relationship

Substrate [ ] and Reaction Rate

• rate of reaction increases as substrate [ ] increases (constant enzyme [ ])

• max activity when enzyme is saturated

• relationship is Exponential
  (levels off when enzyme is saturated)

Reaction Velocity and Substrate Concentration

Low solute [ ] = lower velocity as compared to higher [S]

Enzyme Catalyzed Reaction Rate

Vmax

at high [S] enzymes are fully saturated

KM

tells how much substrate is needed to reach half Vmax

Reaction Calculation for Catalytic Efficiency

Michaelis Menten Equation

Velocity = (Vmax)([S]) / Km + [S]

Lineweaver-Burke Plot

used to analyze enzyme kinetics

Calculation for Kcat

Kcat = turnover number (rate at which substrate is converted to product)

Kcat = Vmax / [E]

Denaturing Enzymes

• damages enzyme

• changes shape & structure

• won’t function properly

  • occurs through change in temp and pH

Enzyme Inhibitors

• chemicals that prevent enzyme from working

• decrease enzyme reaction rate

• can be released

Reversible Competitive Inhibitor

• goes on and off allowing enzyme to regain activity when it leaves 

• reversible & has structure like substrate

• competes with substrate for active site

• reversed by increasing [S]

Competitive Inhibitor Example

Malonate competes with succinate and inhibits succinate dehydrogenase

• can be reversed by adding more succinate

Reversible Non Competitive Inhibitor

• has structure that is different than substrate

• binds to non active site

• distorts shape of enzyme which alters shape of active site and prevents substrate binding

• effect is NOT REVERSED by adding more substrate

Irreversible Enzyme Inhibitors

• inactivates enzyme by bonding COVALENTLY to a particular group in active site

• CANNOT BE REVERSED

• typically ions & heavy metals

Allosteric Regulation

• once there is enough product, production slows down (product acts as its own inhibitor)

• FEEDBACK INHIBITION

Enzyme Activators VS Repressors

• site that is different than substrate binding site

• can function as repressor (inhibitor) or activator

Covalent Regulation

• regulation of enzymes due to addition or removal or specific groups via covalent bonds

• adds or remobes phosphate, methyl or acetyl groups

  • INCREASES REACTION RATE

  • INHIBIT REACTION RATE

  • ADJUST ACTIVITY OF ENZYME

Binary Fission

• cell division in prokaryotes

• occurs at the origin of replication, with the separation of two daughter chromosomes

  • is plausible that mitosis evolved from binary fission

The Par System

• chromosome segregation is due to septum formation in daughter cells

• the Par System ensures equal distribution of chromosome and plasmids in bacteria

Divisome

• Fts proteins interact to form the divisome

  • divisomes are responsible for cytokinesis

Eukaryotic Chromosomes

• numerous origins of replication

• kinetochore connects centromere to spindle apparatus, which is crucial for segregation

• range from 10s to mils of BP

Phases of the Cell Cycle

• consists of

  • mitotic phase (mitosis and cytokinesis)

  • interphase (cell growth and duplication of chromosomes)

• Interphase is divided into G1 (growth phase), S (synthesis) and G2 

• Growth occurs in all of chromosomes but chromosome duplication only occurs in the S phase

• some cells may exit G1 and remain dormant in G0 at a steady state

Eukaryotic Chromosome Specialization

• special sequences in telomeres prevent translocations and shortening

• the centromeric and telomeric sequence are found where repetitive sequences are

Plasmids

• circular

• replicate independently

• gives bacteria competitive advantage

  • antibiotic resistance

Phases of Mitosis

• prophase

• prometaphase

• metaphase

• anaphase

• telophase

Ending G2 Phase

• the nucleus is enclosed by a nuclear envelope that contains one or more nuclei

• chromosomes are not yet condensed and hence not visible individually (first look like spaghetti when not condensed)

Centrosomes

• specialized regions within animal cells that facilitate the organization of microtubules in the spindle

• comprised of a pair of centrioles

Early Prophase

• chromatin fibers undergo tight coiling forming discrete chromosomes that can be visualized

• disappearance of nucleolus

• mitotic spindles begin to form

• aster formation

Replicated Chromosomes

• consists of two identical sister chromatids connected by cohesins

Mitotic Spindles

• composed of centrosomes and microtubules

Asters

• shorter microtubules extending from centrosomes in radial arrays

• anchor and brace for a pull

Centrosome Components

• mother centriole

• distal/subdistal appendages

• proximal/distal ends

• interconnecting fibers

• daughter centriole

• microtubule triplet

Late Prophase (Prometaphase)

• nuclear envelope degrades

• chromosomes are fully condensed

• centrosomes on opposite ends

• spindle fibres are present

Prometaphase (Kinetochore)

• each chromatid has a specialized protein structure called a kinetochore at its centromere

• some microtubules attach to kinetochores which is referred to as kinetochore microtubules

• 46 kinetochores per cell 

Mitotic Spindles

• rock chromosome back and form to slowly move them to the centre of the nucleus

Metaphase

• centrosomes have now positioned themselves at opposite poles of the cell

• chromosomes line up at metaphase plate

• kinetochores of sister chromatids on each chromosome are attached to microtubules coming from opposite plates

Anaphase

• shortest stage of mitosis

• Separase: enzyme tear cleaves cohesins

• allows abrupt separation of chromatids

• as kinetochore microtubules shorten, 2 daughter chromosomes move to app. ends

Telophase

• cell produced 2 daughter nuclei

• nuclear envelope is formed

• reappearance of nucleolus

• chromosomes begin to decondense

• spindle microtubules are deppolymerized

Cytokinesis

• cleavage furrow is crucial step 

• divides cell into 2

  • actin ring squeezes cell

• plant cells are characterized by cell wall — too rigid to divide

  • cell plate divides 2 daughter cells 

Meiosis

• sex cell division

• ends with formation of 4 separate gametes

Prophase I

• chromosomes condense & form pairs

• chromosomes align with homologous partners to match positions on entire length 

Crossing Over

• process where homologous chromosomes exchange parts 

• facilitated by a protein called synaptonemal complex

• linkage of homologous chromosomes at chiasmata

Metaphase I

• pairs of homologous chromosomes align at metaphase plate

• randomization at this stage

Anaphase I

• homologous chromosomes undergo separation & migrate towards app poles of cell

• sister chromatids remain linked & DO NOT DETACH

Telophase I

• homologous chromosomes reach opposite poles of cell 

• some experience reformation of nuclear membrane, others prepare for meiosis II

• cytokinesis forms 2 daughter cells

Prophase II

MEIOSIS

• chromosomes condense & nuclear envelope disintegrates (if needed)

• spindle forms between centrosomes

Metaphase II

MEIOSIS

• microtubules from app poles capture 2 sister chromatids

• chromosomes align individually along metaphase plate 

Anaphase II

• sister chromatids are divided & polled towards opposition poles of cell 

Telophase II

• nuclear membrane envelopes each chromosome

• cytokinesis segregates chromosome sets into new cells

• 4 haploid cells each with a single chromatid

Gametes

• haploid cells produced by gametogenesis

• defines gender as male or female

• male produced sperm (small & mobile)

• female produce ova (immobile, large, nutrient dense)

Fertilization

• process of sperm & egg fusion, leads to creation of diploid zygote 

Genes & Traits

• traits of an organism are a result of molecular expression within the cells

4 Levels of Biological Organization

• Molecular level (functioning pigment of enzyme)

• Cellular level (lots of pigment, little pigment)

• Organism level (dark or light butterfly)

• Population level (dark butterflies in forested regions, light in unforested

Mendelian Inheritance

• concluded that traits appear & vanish in diff generations

• observed trait is dominant & masked trait is recessive

Mendelian 4 Rules

1. nature is full of variation — constant changing

2. tracking genes across generations requires observable variation

3. genetic laws account for inheritance of variation

4. Mendel’s laws are applicable to energy sexually reproducing organism

Homozygous & Heterozygous

Homozygous: carry same alleles

Heterozygous: carry different alleles

Genotype & Phenotype

Genotype: organism’s alleles

Phenotype: outward expression of alleles

Sex-linked genes

• uncovered by Thomas Hunt Morgan in 1909

• gene located on either sex chromosome is a sex-linked gene 

Exceptions to Mendel’s Rules

• no definitively dominant or recessive allele

• 2 or more genes can be linked on same chromosome

• multiple genes involved

• mitochondrial inheritance

• gene-environment interactions

Incomplete Dominance

• heterozygous phenotype is intermediate between 2 homozygotes

Codominance

• 2 alleles for each autosomal gene

• both alleles are expressed