Biochemistry Exam 3

Enzymes

biological catalysts that increase biochemical rxn rates

A substance produced by a living organism that acts as a catalyst to bring about a specific biochemical rxn

• can increase rates 10^6 - 10^14 times faster than if not present

James Sumner (1926)

Discovers enzymes are proteins

• isolated urease from jack beans

• Purified and crystallizing the enzyme

John Northrup (1929)

Purifies pepsin

• digestive enzyme that work optimally in stomach acid (low pH)

• cleaves peptides into smaller peptides during digestion

• Won Nobel Prize in 1946, for “preparation of enzymes in pure form”

Lock and Key Model of enzyme specificity

• 1st proposed by Emil Fischer in 1894

• High-specificty of enzyme-mediated catalysis is best explained by rigid

• best working model until 1958

• Problems with model: could not explain

  • how enzymes are regulated

  • how substrates bind to sites buried deep within the interior of the enzyme

David Kashland (1958)

• Proposed “induced fit model” of enzyme catalysis

• enzyme is analogous to a “glove”

• glove has a 3D shape but is able to be flexible

• can accomodate a flexible hand (substrate)

• enzyme flexibility is able to accomodate the “ill-fitting” subbstrate

Advantages of this model:

1. Permits larger number of weak interactions between enzme and substrate to occur

2. small structural adjustments in enzyme upon binding

Critical Aspects of Enzyme Structure and Function

1. Enzymes usually bind substrates with high affinity and specificity

2. Substrate binding to the active site induces structural changes in the enzyme

3. Enzyme activity is highly regulated in cells

Catalyst

A substrate that increases rate of chemical rxn without itself undergoing any permanent change

Enzymes are catalysts

alter rates of rxn without changing the ratio of substrates & products @ equilibrium

Henry Eyring (1930s)

Transition State Theory

• conversion of a substrate to product involves a high energy transition state where a nucleotide can either become a product or remain as a substrate

• Transition state is very unstable! lasts less than 10^-15s

Reaction Coordinate Diagram

Catalysts lower the amount of energy required to reach the transition state (the activiation energy)

Malate Dehydrogenase

• important enzyme in TCA cycle

• catalyzes the conversion of malate to oxaloacetate

What do enzymes often require to aid in their catalyst rxn mechanisms?

Cofactors

Cofactors (metals)

small molecules that aid in the catalytic rxn within the active site

• an enzyme bound to a cofactor = haloenzyme (“active form”)

• an enzyme without a cofactor = apoenzyme (“inactive form”)

• examples: Fe²+. Cu²+, Mg²+

Coenzyme

enzyme cofactor that require organic components (include many vitamin derived species)

Prosthetic Groups

coenzyme permanently associated with enzyme (covalently attached)

1 Oxidoreductase

oxidation-reduction, transfer of H or O atoms, example: Oxidases

2 Transferase

transfer of functional groups, example = kinases

3 Hydrolase

formation of 2 products by hydrolyzing a substrate, example = lipases

4 Lyase

cleavage of C-C, C-O, C-N bonds without using H2O or oxidation, example = carboxylases

5 Isomerase

Intramolecular rearrangements, transfer of groups with molecules, example = muctases

6 Ligase

formation of C-C, C-O, C-S, or C-N bonds, require ATP cleavage, example = Synthetases

In a chemistry setting, increasing temperature, pressure, and substrate concentration does what?

It increases the likelihood of molecule collisions and reaction rate

In cells, decreasing the substrate concentration @ ambient temperature and 1 atm does what?

It does nothing, it’s not conductive to chemistry happening, but nature overcomes these limitations by using enzmyes

Name the 3 ways that enzymes increase the rate of rxn in cells?

1. They decrease the activation energy by stabilizing the transition state

   • lowers the activation barrier

2. They provide an alternate path to product formation

   • could involve the formation of a sbale rxn intermediate(s) that are covalently attached to the enzyme

3. They orient the substrates appropiately for the rxn to occur

   • proper coordination , decrease entropy of the rxn

3 Common Catalytic Rxn Mechanisms

1. Acid-Base Catalysis

2. Covalent Catalysis

3. Metal-ion Catalysis

Enzyme Reaction Mechanisms

• In general, many enzymes contain a catalytic triad @active sites

• 3 residues from an H-bonding network

  • The H-bonding network is the ideal orientation to help catalyze the reaction

• serine proteases (enzymes involved in protein digestion) - family of endopeptidases (cleaves without a polypeptide sequence)

• all contain a catalytic triad

Chymotrypsin

specifically cleaves after aromatic residues except when proline is after

• i.e. F, Y, W

• incolves acid-base & covalent catalysis steps

Trypsin

specifically cleaves after positively charged resides except when proline is after

• i.e. R, K

Elastase

specifically cleaves after small hydrophobic residues except when proline is after

• i.e. A, V, S, G

What are the essential roles of nucleotides in biology?

1. Energy currency in metabolic processes (ATP, GTP)

2. Intracellular signaling in response to hormones / extracellular stimulus (ex. insulin, histamine)

3. Important structural component of cofactors & metabolic intermediates (Acetyl-CoA, NAD^+, FAD)

4. Basic building blocks of nucleic acids

   DNA - deoxyribonucleic acid

   RNA - ribonucleic acid

3 main characteristics of nucleotides

1. A nitrogenous (N-containing) base, purines (bigger) & pyrimidines (smaller)

2. A pentose (5-membered beta-furanose ring), sugar and carbohydrate

3. 1 or more phosphate groups

Nucleoside

no phosphate present, just base + ribose

Nucleotide

base + ribose + phosphate

Ribose Puckering

• endo = towards base

• exo = away from base

• important implications for 3D structure of DNA

• 2’ endo & 3’ endo are preferred in B-DNA (most favorable state)

• 3’ ends twist in RNA

5-methylcytidine

Different type of nucleoside or nucleotide

• this is how bacteria differentiate between self and foreign DNA

• defense mechanism

• we use enzymes that target this when making new molecular clones

Phospodiester bonds (in both DNA and RNA)

• nucleotides covalently linked together by phosphate group “bridges”

• The 5’ phosphate of one nucleotide covalently attached to 3’ OH of next nucleotide, always goes from 5’ end to 3’ end

• Made of alternating ribose and phosphates

• phosphate = - charge, bind to + charged proteins and + metal ions

• OH bonds interact with water

• Mg is added to prevent negative charge so there is no repulsion issues

How is DNA more stable than RNA

RNA lacks the OH on the 2nd carbon

Describe the Hierarchy of DNA & RNA

1. covalent structure and nucleotide sequence (5’ AGCT.. 3’)

2. any regular stable structure by some or all of the nucleotides in the sequence (Helix)

3. higher order structures of DNA & RNA

Friedrich Miescher (1868)

isolated and characterized a phosphorus-containing substance he called “nuclein”

Avery, MacLeod, and McCarty (1944)

injected virulent DNA from S. pneumoniae (pneumonia) into a non-virulent strain, and observed the virulent change

Chase and Hershey (1952)

• infection of bacteria by virus (bacteriophage) with radioactively labeled DNA or protein

• proved that DNA carried genetic information

Chargaff (1940s)

• 4 nucleotides (A, T, C, G) occur in different ratios in different organisms

  • amounts of certain bases are closely related

Chargaff’s Rules

1. Base composition of DNA generally varies from one species to another

2. DNA specimens from different tissues of the same species have the same base composition

3. Base composition of DNA doesn’t change with an organism’s age, environment, or nutrition

4. Regardless of species, the # of A = # of T, and the # of C = # of G

   • therefore, sum of pyrimidine residues equals the sum of pruine residues. A+G =T+C

Wilkens and Franklin

X-Ray studies

1. Found the DNA molecules are helical

2. 2 periodicities in helix (3.4 A and 34 A)

Key Features of Watson & Crick DNA Model

1. Right-handed double helix

2. hydrophilic backbones of alternating deoxyribose and phosphate groups on outside of helix (for interactions with H2O)

3. Furanose rings of each deoxyribose are C2’ endo config

4. Purine & Pyrimidine bases of both strands are stacked inside double helix

5. Offset pairing creates major and minor grooves

6. Each nuclotide base of 1 strand is paired in the same place with a base on the other strand

7. H-bonded G-C and A-T fit best within structure, follows Chargaff’s Rules (Mechanism)

8. Antiparallel orientations for each strand

9. 36 A periodicity of helix, 10.5 bp per turn

Forces holding DNA double helix together

• H-bonding between complementary base pairs = specificity

• Base stacking, non-specific but important for stability

Replication of DNA by…

1. separating 2 strands (parent strands), act as a template because A-T & C-G

2. synthesis of copies (daughter strands)

B-form DNA (Watson-Crick)

• right-handed

• double-stranded DNA

• well hydrated

• “relaxed DNA”

A-form DNA

• right-handed

• favored in solutions without H2O, preferred in RNA but function is unknown

• base pairs are not perpendicular to helix axis (tilted by 20 degrees)

• more squat: major grooves are deeper & minor grooves are shallower

Z-form DNA

• left-handed helix

• purines are in syn-config

• base pairs are not perpendicular

• more slender & elongated: major grooves are not apparent & minor grooves are narrow and deep

• observed in bacteria & eukaryotes, function is gene regulation and gene recombination

Describe a palindrome

• reads the same fowards and backwards

• has 2-fold symmetry

• has ability to form 2° structures

Mirror repeat

assuming it repeats but can’t form 2° structure

Talk about single stranded DNA & RNA with palindromes

• can form hairpins

• important for:

  1. mRNA (causes RNA polymerase to fall off, ending transcription)

  2. RNA 2° structure (ribosomes, tRNAs, ribozymes)

  3. restriction enzymes, used for analysis of DNA cloning & recombinant DNA manipulation

Restriction Enzymes

defense mechanisms within that cleaves DNA and RNA, but shows up 0 times bc its for defense

Types of Restriction Enzymes

1. EcoRI - from E.coli, 4-base overhang (sticky)

2. SmaI - from s.marcesans, blunt (straight cut), no overhang

3. NdeI - from N.denitnificaas, 2-base overhang (sticky)

Talk about RNA World Hypothesis

• self-replicating RNA molcues were precursors for current life on Earth

• DNA (storage, and more stable than RNA) leads to RNA (mRNA, tRNA, rRNA) leads to Protein (enzymes)

• RNA is @ important junction, predates RNA

Bacterial gene organization

monocistronic: 1 gene in mRNA molecule

polycistronic: more than 1 gene in mRNA molecule

Hairpin @ the end, stalls RNA polymerase, causes it to fall off

Describe different types of RNA

mRNA - specific instructions copid from DNA into RNA for ribosome to read and link a.a. together (protein synthesis)

tRNA- transfer RNA, takes a.a. to ribiosome for protein syntheis

rRNA- ribosomal RNA, building blocks of ribosomes

Nucleic Acid Chemistry

• protection & maintenance that is integral to keeping DNA stable and unaltered

• necessary strand separation for DNA replication transcription

DNA denaturation

1. Increasing temperature - weakens H-bonds & unzips DNA

2. Increasing pH - deprotonation of guanine and thymine

3. Ionic strength - decreasing salt concentration causes repulsion of negative charges in backbone

   • A higher salt conc woulf protect from negative charge of phosphate

Describe Non-enzymatic transfromations

1. Deanimation - loss of exocyclic group

   • 100x more often for C to U

   • cell machinery can tell difference between U and T and fixes it

2. UV Light - condensation of 2 ethylene groups to form a cyclobutane ring

   • why we need to wear sunscreen

What are the outcomes if a cell can’t fix the non-enzymatic transfromations?

1. Aging (oxidative stress, mistakes from replication)

   • decoupled e- from ETC, leads to oxidation of: deoxyribose, bases (A T C G), and strand break (Worst one) (SOD mechanism)

2. Carcinogenesis (Cancer)

   • UV damage, mistakes during replication

Primer

Short strand of nucleic acid sequence (10 to 15 bases) that serves as a starting point for DNA synthesis

• must have a a free 3’ OH

• needed for Sanger Method for DNA Sequencing (Mechanism)

What are NTPs and dNTPs

Energy currency of the cell

• makes things thermodynamically favorable

• coupled rxns with ATP hydrolysis