Biolchem 212 Exam 2 UMich 2023 Memorize These!

Functional Groups

Ammonium ion, amine group: -NH3+, -NH2

Hydroxyl group: -OH

Carbonyl group: O=C--

Carboxyl group, Carboxylic Acid: O=C--OH, O=C--O_

Amide group: O=C--N--H

Thiols: -SH

Sulfides: -S-

Disulfides: -S-S-

Functions of Proteins

Enzymes: catalyze biochemical reactions

Hormones: messenger that regulate bodily functions

Storage Proteins: store essential substances for immediate use

- Ovalbumin (egg whites)

- Casein (milk)

- Ferritin (stores iron)

Transport Proteins: carry substances through blood & through the cell membranes

Structural Proteins: supports & maintain shape of cell

- Collagen: support for bones and teeth

- Elastin: ligaments

- Keratin: fingernails

Protective Proteins: provide defense against invaders & protect from injury

- Antibodies: recognize foreign substances

Contractile Proteins: do mechanical work

- Actin & Myosin: Muscles

- Kinesin: Vesicle Transport

Four classes of Amino Acids

Nonpolar, Neutral Polar, Acidic, Basic

Chiral Carbon

A carbon atom attached to four different atoms or groups of atoms.

Achiral Carbon

Can be rotated on mirror image. Two functional groups are the same.

Isomers

Compounds with the same formula but different structures.

Constitutional Isomers: Isomers that differ in the bonding order of atoms (ex: H3C-CH2-OH H3C-O-CH3)

Stereoisomers: Isomers that have atoms bonded in the same order, but with different arrangements in space (cis-trans)

Stereoisomers

Isomers that have atoms bonded in the same order, but with different arrangements in space

Enantiomers: mirror images of stereoisomers that cannot be superimposed

Diastereoisomers: stereoisomers that are not mirror images

Nonpolar Amino Acids

Side chains are alkanes and arenes (no charge)

Alan Is a Methhead he Tries to Violate Leucin he is Going Pro in Fetnal

Ala, Ile, Met, Trp, Val, Leu, Gly, Pro, Phe

Arenes: aromatic hydrocarbon with alternating double carbon bonds in ring

Neutral Polar Amino Acids

Side chains interact with water (no charge)

Serine Throws Cystine Trying to Ascend into Greatness

Ser, Thr, Cys, Tyr, Asn, Gln

All have hydrogen bond donors and accepters but Cys

Acidic

Side chains with a negative charge

Aspirin and Glucose are negatively impacting society

Asp and Glu

Basic

Side chains with positive charge

Lions Argue over His land

Lys, Arg, His

Zwitterion

A molecule that contains charges, but is neutral overall. Most often used to describe amino acids. All amino acids are zwitterionic at pH 7

Peptide Bond

Two amino acids covalently linked together by an amide bond. An amide bond that joins two amino acids is a peptide bond.

Shape Determining Interactions

Covalent Bonds:

-Intramolecular Bonds (Amino acids, peptides & disulfides)

-Disulfide Bonds: form covalent bonds that stabilize protein structure once it's folded in tertiary structure

Noncovalent Interactions:

-Electrostatic Interactions (Salt bridges/ Ionic attractions)

-Hydrogen Bonds: occur between H-bond (O-H and N-H) and acceptors (O and N)

-Van der Waals Forces

-Hydrophobic Effect: side chains of nonpolar residues exclude water to pack together in center of protein

Structural Amino Acids

Cystine is a Pro at Gliding

Gly: no organic side chain, flexible

Pro: secondary amine forms tertiary amide upon formation of peptide bond, makes a ring shape.

Cys: thiol (-SH) group forms disulfide bonds in extracellular proteins, binds metals.

Insulin

A hormone produced by the pancreas or taken as a medication by many diabetics.

Primary sequence consists of 2 polypeptide chains held together by 3 disulfide bonds.

Preproinsulin > Proinsulin > Insulin

Secondary Structure: a-Helix

Right-handed coil resulting from hydrogen bonding; common in fibrous structural proteins

3.6 residues held together by amide groups

Stabilized by hydrogen bonds between peptide bonds

1) form peptide bonds between adjacent residues

2) hydrogen bond between amide groups 4 residues away

Secondary Structure: B-Helix

B-pleated sheets are made of B-strands

A B-pleated sheet is held together by amide groups

1) form peptide bonds between adjacent residues

2) hydrogen bond between amide groups on adjacent strands

Protein Structure Classification

Globular: forms a globe-like structure, typically water soluble as hydrophilic residues are on the surface

Fibrous: forms fibers or sheets, insoluble small hydrophobic residues packed tightly together. All a-helical or B-sheet

Conjugated Proteins

Require non-protein components for function

Greek Letters Make Pronunciation Hell for Newborns

Glycoproteins: covalently bound carbohydrates (receptors on cell surface)

Lipoproteins: covalently bound to lipids (transport cholesterol through the blood)

Metalloproteins: metal ions (trace minerals) for structure stabilization or catalytic function (ex: zinc)

Phosphoproteins: covalently bound phosphate groups used to activate or deactivate a protein or provide binding sites for protein attachment.

Hemoproteins: contain heme groups/porphyrin rings (ex: oxygen transporters, drug metabolizing enzymes)

Nucleoproteins: contain ribonucleic acids (ribosomes used in protein synthesis)

Oxygen Transfer

At lower concentrations of oxygen (as in the capillary), myoglobin has higher affinity for oxygen that does hemoglobin

Sickle Cell Anemia

Genetic disorder, acidic residue is changed to a hydrophobic residue. Globular protein becomes fibrous protein.

Electrophoresis

Is a technique used to separate proteins based on their charge and molecular weight.

Fibrous Proteins: Collagen

Tough, Insoluable, Triple helix

Residues: Gly, Pro, and Thydroxlated Pro (made from Vit C)

Connective tissue, Bones, and Teeth

Collagen Related Diseases

Scurvy: Vit C deficiency

Brittle Bone Disease:

- Type 1: normal collagen, just insufficient amounts

- Type 2: poor amounts and quality of collagen

Can accidentally be seen as child abuse

Fibrous Protein: Keratin

- Found in hair, skin, nails

- Predominately secondary structure: alpha helix

- Stabilized by high number of disulfide bonds

- Rich in cysteine

Hair curls with heat because heat breaks noncovalent bonds

Hair curls with perm because chemicals break disulfide bonds

Chaperones

Proteins that assist in protein folding keeps proteins from aggregating in nonfunctional units

Shakes until protein is folded

Prions

Misfolded proteins

Proteins in CNS (brain)

Convert a-helices to B-sheets

Induce other normally folded proteins to change shape

Transmissible Spongiform Encephalopathies (TSE)

Progressive brain infections caused by unusual pathogens called prions, leading to loss of brain structure and function.

Protein Degradation

Autophagy: self-degradative process, how prions die they kill themselves

Lysosomes: recycle center, acts as garbage disposal

Proteasomes: degrade unfolded or unneeded protein by proteolysis

Protein degradation occurs in proteasomes, lysosomes, and digestive tract

Protein Denaturation

He is a Mechanic, he uses Detergent On Parts In cars

Heat: disrupts the non-covalent interactions

Mechanical Agitation: foaming/air bubbles cause lack of water

Detergents: interact with hydrophobic side chains and unfold the protein

Organic Compounds: polar solvents can disrupt H-bonds

pH Change: basic and acid side chains change charge disrupting salt bridges

Inorganic Salts: at high concentrations and disrupt salt bridges

Anatomy of Biological Reactions

Enzyme: bio catalyst, "ase"

Substrate: reactant of enzyme

Turnover number: # of substrate converted to product over a time period

Cofactor: nonprotein binds to protein required to catalysis

Coenzyme: organic cofactors typically found to Redox enzymes (like vitamins)

Apoenzyme: protein - cofactor/coenzyme

Holoenzyme: catalytically active enzyme containing apoenzyme with cofactor/coenzyme

Active site: pocket in enzyme where catalysis takes place

Catalytic Role of Enzymes

1. Proximity effect (bring reactants tg)

2. Orientation effect (hold reactants at required distance and orientation for reaction)

3. Energy effect (lower AE by inducing a strain of the bonds of the substrate)

4. Catalytic effect (provide acidic, basic, and other types of functional groups that are required for catalysts)

6 Enzyme Classes

1) Oxidoreductase: catalyze redox (e.g. alcohol dehydrogenase) subclass: dehydrogenases

2) Transferase: transfer of functional groups between substrates subclass: kinases (transfers phosphory) transaminase (transfers amino groups)

3) Hydrolases: catalyze hydrolysis of covalent bonds subcalsses: lipase, proteases, nucleases, amylases

4) Isomerases: catalyse intramolecular rearrangements

5) Lyases: catalyze elimination/addition of functional groups around a double bond subclasses: decarboxylases, dehydrases, deaminases, synthases

6) Ligases: catalyze bond formation couple with ATP hydrolysis subclasses: synthetases, carboxlases

Rules for Oxidation

1. loss of an e-

2. loss of a covalent bond to H

3. gain of a covalent bond to O

4. reducing agent

Rules for Reduction

1. gain of e-

2. gain of a covalent bond to H

3. loss of a covalent bond to O

4. oxidizing agent

Induced Fit

The change in shape of the active site of an enzyme so that it binds more snugly to the substrate, induced by entry of the substrate.

How an Enzyme Works (5 steps)

1. Enzyme is available with empty active site

2. Substrate binds to enzyme

3. Water is added

4. Substrate is converted to products

5. Product is released

Factors Affecting Enzyme Activity

- Temperature: if you heat proteins you break noncovalent forces (37 c is the max rate before declining)

- pH: max enzyme energy is appox a pH of 7

- Enzyme Concentration: enzyme amount increases, excess substrate, no max

- Substrate Concentration: enzyme amount doesn't change, rate approaches a max speed

Enzyme Inhibition

- Competitive Inhibitor: resembles substrate and competes for same binding site on enzyme. Vmax unchanged Km increased

- Noncompetitive Inhibitor: structure of inhibitor doesn't resemble the substrate and binds to a separate site on the enzyme causing a change in active side configuration, in turn, preventing the substrate from binding. Vmax lowered Km unchanged

- Uncompetitive Inhibition: doesn't resemble the substrate and binds to the enzyme substrate complex, but not at the active site. It can't bind the enzyme alone. Vmax and Km lowered

- Irreversible Inhibition: covalent modification of enzyme rendering it inactive (ex: aspirin)

- The lower the Km, the greater the enzyme's affinity (want) for the substrate

Enzyme Regulation

- Allosteric Regulation (+ and -): substrate binding at the active site is effected by the binding of a molecule at a different site (ex: noncompetitive inhibitor)

- Feedback Control (+ and -): a product in a metabolic pathway is an allosteric regulator of an enzyme that catalyzes a critical step in that pathway

- Covalent Modification: add phosphate group on tyrosine, serine, or threonine can trigger conformational change in structure (activates/deactivates)

- Genetic Control: gene for an enzyme is turned off or on to regulate the amount of enzyme present in the cell

- Zymogens: enzyme is synthesized as a proenzyme (inactive form) cuts a protein segment to activate. (ex: digestive enzymes)

Vitamins

- Organic molecules essential to biological functions

- Water Soluble: hydrophilic contain OH & COOH, vit C, vit B

- Fat Soluble: hydrophobic containing mainly carbons and hydrogens, vit A, D, E, K

Fat Soluble Vitamins

- Vitamin A: sight, provit B-carotene, active retinol

- Vitamin D: bone & teeth (sun), precursor 7-dehydrocholesterol, active calcitriol

- Vitamin E: antioxidant (prevent clotting), active a-tocopherol

- Vitamin K: form blood clots, active K1/K2

Water Soluble Vitamins

All water soluble vit are coenzymes

B1: Thiamine, TTP, metabolism

B2: Riboflavin, FMN, FAD (oxidized)/FADH2 (reduced), redox

B3: Niacin, NAD+ (oxidized)/NADH (reduced), NADP+ (oxidized)/NADPH (reduced), redox

B5: Pantothenic Acid, CoA, carrier of acetyl groups (2 carbon)

B6: Pyridoxine (amine, alcohol, aldehyde), CoPLP (gains phosphate group), amino acid metabolism

B7: Biotin or Vit H, CoBiotin or Biocytin, carbon carrier in carb and lipid metabolism and forms amide bonds with amine groups on proteins

B9: Folic Acid, CoTatrahydrofolate, carry 1 carbon in form of heme rings, protects from cardiovascular disease, chemotherapeutic

B12: Cobalamin, CoMethylcobalamin, methyl transfer, nucleic acid metabolism, intestinal bacteria

Vitamin C: L-ascorbic acid, CoC, antioxidant, energy production, disease prevention, collagen formation

Antioxidant = reducing agent

Diagnostic Enzymes

Liver (hepatitis) & Heart Attack: AST ALT LDH

Heart Attack: CK or CPK

Liver (alcoholism): GGT

Liver Dysfunction: GPT

Rickets & Carcinoma: ALP

Isozymes

Two enzymes that can catalyze the same reaction

Rapid Covid-19 Test

Analyte > Antibodies conjugated Tag (to show color) > Capillary Flow > Test Line (Antibodies) > Control Line

Nucleotides

Basic units of DNA molecule, composed of a sugar, a phosphate, and one of 4 DNA bases

Phosphate + Nucleoside

Hydrogen Bonding

Acceptor: O or N with lone pair

Donor: O of N directly bonded to H

Amide Formation

Carboxylic Acid + Amine > Amide + Water

Nucleoside

Sugar + Base

Multiple Phosphates

All negative charges

Monophosphate: AMP

Diphosphate: ADP

Triphosphate: ATP

d: lost oxygen

Difference between RNA and DNA

RNA has an extra O

Stacking Energy

The energy of interaction that favors the face-to-face packing of purine and pyrimidine base pairs.

Protein-DNA Interactions

Basic amino acids (Arg, Lys, His) interact via salt bridges with negatively charged phosphate backbone

Semiconservative Replication

Method of DNA replication in which parental strands separate, act as templates, and produce molecules of DNA with one parental DNA strand and one new DNA strand

RNA Transcription

DNA > RNA

DNA Coding Strand = mRNA (T = U)

DNA Noncoding Strand = 3'-5'

Protein Translation

DNA > transcription > mRNA > tRNA > translation > Protein

Nucleic Acid Synthesis

Phosphoanhydride hydrolysis provides the energy, it's always spontaneous (exergonic)

PPi (Pyrophosphate) hydrolysis is considered irreversible

DNA Polymerase Reaction (Replication)

The enzyme that catalyzes the synthesis of new DNA (replication) (2 ATP)

dNTP > dNMP

DNA Polymerase adds nucleotides onto the 3' OH of nucleic acid (primer strand)

Replication Initiation

Helicase: unwinds DNA double-helix by hydrolyzing ATP

Topoisomerase: Removes strain by hydrolyzing ATP

Single-stranded binding protein: coats single-stranded region of DNA so that double helix doesn't re-form

Primase: synthesizes primer made of RNA from which DNA polymerase can build new strand

AT bare-pairs

Replication Bubble

Replication Elongation

DNA polymerase only synthesizes 5' > 3'

Leading strand: continuous

Lagging strand: discontinuously, okazaki fragments

DNA clamp: promotes processivity

Proofreading: DNA polymerase can "check" the last base it added and remove it with 3' > 5' exonuclease. Checks size and H-bond acceptor location of base-pair in minor groove

Replication Termination

DNA polymerase reaches RNA primer, the primer is pushed out of the way and removed by endonucleases

DNA ligase: okazaki fragments are separated by nicks and joined into a single strand by DNA ligase, reaction uses PPi

Telomeres: active in dividing cells, repetitive DNA sequences at the end of linear chromosomes, extended by telomerase (RNA dependent)

Types of RNA

mRNA: product of transcription that carries info for translation

tRNA: carries amino acids to ribosome for translation

rRNA: what makes up a ribosome, the catalyst for translation

RNA Polymerase

enzyme that catalyzes the synthesis of RNA from a DNA template (PPi)

a hydrid helix between DNA template strand and RNA product forms inside RNA polymerase

Transcription Initiation

RNA polymerase attaches to the promoter region of DNA strand, and synthesis begins

Transcription factors bind to promoter sites; helicase unwinds DNA, DNA must be unpacked (removed from nucleosome)

Add acetyl group to his and lys removes positive charge

Transcription Elongation

Phosphorylation of RNA polymerase allows for elongation, phosphorylation disrupts interactions between RNA polymerase and transcription factors bound at promoter.

Transcription Termination

A signal in mRNA leads to disruption of RNA polymerase

RNA-dependent: RNA product forms short helix that disrupts RNA polymerase

Protein-dependent: Protein (Rho) binds a sequence in RNA product and disrupts RNA polymerase

mRNA isn't ready for translation until they have been spliced

Spliced: exons are translated, introns are removed

Amino Acid Loading of tRNA

3' OH of tRNA is linked to carboxylic acid of amino acid to form an ester (PPi)

Ribosome

Contains both protein and rRNA

Brings together mRNA and tRNA during translation

A-site: amino acyl tRNA binding site

P-site: peptidyl-tRNA binding site

E-site: exit site, deacylated tRNA

Translation Overview

Protein chain is attached tRNA in P site

Amino group of amino acyl-tRNA in A site attacks ester of tRNA-AA linkage in P site

New amide bond formed

Protein chain is now attached to tRNA in A site

Translation Initiation

Small subunit binds mRNA and initiation factors

Initiator tRNA binds at start codon

Start codon is usually AUG (Met)

Large subunit binds with initiator tRNA in P site

GTP hydrolyzed to GDP to release initiation factor (Pi)

Translation Elongation

1. Decode (check correct codon-anticodon) GTP > GDP + Pi

2. Transpeptidation (make new peptide bond)

3. Translocation (move down one spot) GTP > GDP + Pi

3 steps, 2 ATP equivalent

Translation Termination

Codon in A site is stop codon, release factors bind and allow water in to hydrolyze ester linkage between protein and tRNA in P site 1 GTP hydrolyzed to 1 GDP to separate all the pieces

1 ATP equivalent

Mutation examples: Cystic Fibrosis & Sickle-cell Anemia

Cystic Fibrosis: deletion

Sickle-cell Anemia: point mutation (SNP)

Polymerase Chain Reaction (PCR)

A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides. 2^n

DNA Sequencing

The process of determining the precise order of nucleotides within a DNA molecule

Use same in vitro DNA Replication

Recombinant DNA

Uses: GMO to fight against bad diseases in crops

DNA produced by combining DNA from different sources.

Plasmid: circular DNA that can be propagated inside bacteria

Restriction Endonucleases

Cas 9

Enzyme that recognizes guide RNA and binds here and cuts the DNA, molecular scissors

RT-PCR test

Same as PCR but with an added step of reverse transcription of RNA to DNA

COVID-19 Tests

Antigen test tend to have more false negatives

PCR takes longer to come with results