What is biochemistry?

• Identify core macromolecules & their basic characteristics:
  • Proteins
  • Nucleic acids
  • Lipids
  • Carbohydrates

• Identify types of experimental strategies used to study them
  • Structural
  • Biophysical 
  • Mass spec
  • Various cell & molecular biology techniques
    • Concept of “reporters”
    • Types of probes

Biochemical intuition & perspective

• Cell Biology by the Numbers
  • Back of the envelope estimates
  • Sense of scale
  • Intuition for concentrations & speeds
• Appreciate that cells are compartmentalized and things like scaffolding proteins and phase separation play a key role in raising local concentrations of molecules involved in a pathway
  • i.e. these conditions may differ greatly from those in bulk solution
• Appreciate that much of biochemistry is probabilistic
  • We can predict whether things will be likely or unlikely, but ultimately it’s a numbers game
    • Even very unlikely and/or rare things can happen to a small degree
  • Especially keep this in mind when thinking about pKa & pH

Foundational chemistry of biochemistry

• Key elements to focus on – H, C, N, O, P, S
• Identify key functional groups – carboxyl, amine, amide, ether, ester, acetyl, phosphoryl
• Keep an eye out for common nucleophiles & electrophiles
• Refresh knowledge of oxidation & reduction
  • Understand why oxidation often (but not always) involves gain of bond to oxygen and/or loss of bond to hydrogen & why reduction often (but not always) involves loss of bond to oxygen and/or gain of bond to hydrogen
• Refresh knowledge of diffusion, osmosis & tonicity, colligative properties

Thermodynamic basics – more detailed coverage later in course; here, emphasis on intuition

• Thermodynamics vs. kinetics
• Dynamic equilibrium
• Gibbs free energy & ΔG
  • Negative vs. positive ΔG
  • Intuition as to what is favorable vs. non-favorable
  • Intuitive sense of scale of ΔG’s for core biochemical reactions
• “High-energy bonds”
  • Intro to role of phosphate groups – be on the lookout for them in reactions and metabolic pathways & predict what role they’re playing
• Reaction coupling – making the non-favorable possible
• Stay tuned for ENZYMES!

Types of intermolecular interactions

• VDW/London dispersion forces/hydrophobic interactions
• Dipole-based
• H-bonds
• Ionic

Water (and why it complicates a lot of things you learned in o-chem!)

• Key characteristics
• H-bonds
• Hydrophobic effect (water exclusion effect)
• Roles of water in biochemical interactions & reactions
  • Direct & indirect involvement in reaction mechanisms
  • Roles in binding energies – entropy upon displacement, etc.

pH 

• appreciate that it’s a log10 scale, so a difference of 1 pH unit might sound small, but it’s really a 10-fold difference
• acids & bases – emphasis on those used in body & lab
• autoionization of water
• pKa
  • rough pKas to remember:
    • carboxylic acid: 4-5
    • protonated amine: 9-10 
    • thiol: 10
    • alcohol: 16-18
    • phosphoryl group: ~2 for first deprotonation, ~7 for second deprotonation

pH buffers

• Henderson-Hasselbach equation
  • be able to calculate how much is protonated vs. non-protonated at a certain pH (will come in handy when learning about amino acids)
  • be able to calculate the amount of an acid & its conjugate base to mix to get a certain pH (will come in handy when preparing buffers in lab)
• Key buffers used in body & lab
  • Bicarbonate buffer system
  • Phosphate buffer system

Overview of 4 levels of protein structure (refer to as you build up from amino acids to protein complexes)

• 1°: sequence of amino acids
• 2°: structural motifs involving backbone interactions (β-strands, α-helices, turns, etc.)
• 3°: overall structure of a single chain including structural features formed by interactions involving side chains
• 4°: structure of a protein involving multiple chains

Amino acids

• Be able to draw core amino acid backbone, complete with stereochemistry
  • Tell apart L vs D (I recommend playing around with molecular modeling kits (e.g. molymod kits))
  • Appreciate that amino acids are almost always in their zwitterionic form, so draw them that way! (amino group protonated & carboxyl group deprotonated)
• I am not a fan of rote memorization, but this is one area where I do think memorization is important…
  • Memorize structures (ideally) or at least be able to identify structures
  • Memorize 1-letter & 3-letter abbreviations
  • Memorizing those will let you focus on the more important (and fun) stuff – their properties!

• Be able to classify by multiple parameters including:
  • Polarity
  • Hydrophobicity
  • Charge
    • Know relative pKas of ionizable side groups & be able to calculate what proportion of the free amino acid form is on average protonated at a given pH
      • BUT also know that the local context within a protein can greatly influence the actual pKa
  • Size
  • Branched/straight chain
  • Aliphatic vs. aromatic
  • Weirdos – what makes glycine & proline unique?
  • Presence of functional groups
    • And thus, the types of reactions and interactions they might participate in
  • Later in the course, be able to classify by:
    • Essential vs. non-essential (in the dietary sense)
    • Ketogenic, glucogenic, or both

Peptide bonds

• Appreciate the partial double bond character of the peptide bond
• Identify phi & psi angles & what limits them (e.g. steric hindrance)
• Be familiar with Ramachandran plots
• Identify cis vs. trans peptide bonds & explain why trans is almost always favored (hint: steric hindrance again)
• Overview of how peptide bonds are actually formed in cells via translation (It’s not a simple condensation reaction as shown in textbooks, but rather a stepwise process. You don’t need to know the details here, but take a quick look at the process – and return to it later – to get an appreciation of it)

Secondary structure

• Recognize β-strands, β-pleated sheets (β-strands teaming up, either in a parallel or antiparallel manner), α-helices, & turns
  • And see how their characteristic psi/phi angles show up on Ramachandran plots
• Identify the backbone-backbone interactions holding these structures together
• Appreciate the “faces” of secondary motifs
• Identify which side chains are next to each other in space
• Explain why certain amino acids prefer to be in certain motifs & predict which type of motif a given amino acid might prefer
  • Long & flexible tends to prefer α-helix
  • Short & bulky tends to prefer β-strand 
  • Glycine & proline don’t like either
    • Glycine is too flexible so it doesn’t like being confined like that
    • Proline is too rigid, so it doesn’t like those angles
• Appreciate the presence and roles of intrinsically disordered regions (IDRs)
  • scaffolding roles, phase separation, etc.
• Be familiar with the technique of circular dichroism spectroscopy (CD) – don’t worry about details, but know that it can help show the relative proportions of α-helices, β-sheets, & IDRs in a protein

Tertiary structure

• Identify types of interactions holding the structure together
  • And which amino acid residues participate in which
• Be familiar with the concept of “domains”
• Appreciate how amino acids far apart in a primary sequence can be close together in the actual structure
• Predict which amino acids would want to be where in a protein
  • Hydrophobic – inside
  • Hydrophilic – surface/solvent-exposed
• Be familiar with disulfide bonds
  • know where you’re more likely to find them (e.g. extracellular proteins)
  • appreciate how glutathione maintains a reducing environment inside of cells
• Understand how heat, chemical denaturants, & reducing agents can unfold proteins
• Appreciate that proteins may have multiple conformations (shapes) they can switch between (undergo conformational change)

Quaternary structure

• Appreciate how a single protein may contain multiple chains
• Know terminology for multimers (dimer, trimer, homo-, hetero-, etc.)
• Identify types of interactions holding the structure together (sometimes includes disulfide bonds)

Post-translational modifications

• Basics of phosphorylation, glycosylation, lipidation, etc.
  • will return to glycosylation & lipidation later in course when discussing carbohydrates & lipids
• Identify which amino acids can get which type of modification
• Appreciate that these modifications can alter a protein’s shape and/or activity and/or interactions
• Understand that specific enzymes add specific modifications to specific sites & that these enzymes can be regulated

Translation

• Appreciate the overall process (and go into in much more detail if you’re interested and have time, because it’s really cool!)

Protein folding

• In vitro vs. in vivo
  • In vitro:
    • Role of primary sequence – Anfinsen’s hypothesis
• Overview of how proteins fold in cells
  • Idea of folding free energy landscape & how the protein can get stuck in local minima
  • Role of chaperones

• Protein misfolding and its role in disease
  • Be familiar with prions
• Be familiar with biochemical techniques for analyzing folding
  • Chemical & thermal denaturation assays
• Basics of protein structure prediction (e.g. AlphaFold)
  • Including strengths & limitations

pI (isoelectric point)

• Understand what it is
• Be able to calculate the pI of a short peptide given the pKas of the component amino acids
• Be familiar with role of pI in biochemistry experiments
  • IEF (isoelectric focusing)
  • Ion exchange chromatography
  • PAGE

Structural proteins

• Be familiar with concepts of scaffolds (proteins holding things together)
• Appreciate how proteins help shape things

Ligand binding

• Binding kinetics & thermodynamics
  • Understand Kd
    • Derive it to see where it comes from
    • Be able to identify it on graphs
    • Be able to compare Kds and recognize that a higher Kd corresponds to a weaker interaction
  • Be familiar with some biophysical techniques used to measure binding (ITC, SPR, etc.)
    • Describe how to set up an experiment to measure binding & calculate Kd from binding data
      • Will be reinforced in lab
• Be familiar with the potential for cooperativity & how it’s described/reported
  • Hill plots & equation
  • Recognize cooperative binding on a graph
  • Hemoglobin is a great example of this
    • Also know how pH affects hemoglobin binding oxygen (Bohr effect), how BPG affects hemoglobin binding oxygen, etc. 
• Understand the concepts of avidity and affinity

Enzymes

• Appreciate that enzymes catalyze reactions in both directions and do not get used up in the process
• Explain the induced fit hypothesis and the idea of the transition state
• Explain how enzymes lower the activation barrier in reactions
  • Identify some of the ways they do this:
    • Holding molecules together
    • Altering local pH
    • Giving & taking protons or electrons 
    • Providing cofactors
    • etc.
• Be familiar with the EC classification system for enzymes
  • Be able to explain what enzymes of each class do 
  • Be able to give examples of enzymes in each class
• Understand & give examples of acid-base catalysis
• Be familiar with types of cofactors
  • in metabolism section, we will encounter several core cofactors
    • you should be able to recognize what role they play and predict which cofactors might be used for what type of reactions (e.g. NADH for reductions)
• Explain roles metals can play in enzymes
• Classical reaction mechanisms to know:
  • Cysteine protease
• Understand enzyme kinetics
  • Get comfortable doing calculations involving Michaelis-Menten kinetics
    • Including units
  • Derive the Michaelis-Menten equation at least once to get an appreciation for it
  • Explain the mathematical definition – and more importantly, the practical meaning – of Km, Kcat, Vmax, and specificity constant (km/kcat)
  • Be able to interpret a Lineweaver-Burk plot (double-reciprocal plot)
    • And also know its
      • Strengths: easy to get values for Vmax & identify types of inhibition; don’t need fancy software to fit curve
      • Limitations: uneven weighting of points with more emphasis on measurements from lower values skews graph
  • Compare and contrast Km and Kd
  • Distinguish between pre-steady-state & steady-state kinetics
    • Describe how to set up an experiment to measure various kinetic parameters
      • Will be reinforced in lab
• Explain the terms “activity” and “specific activity”
  • Appreciate how specific activity should increase as an enzyme-containing solution become more pure
• Appreciate how an “Activity Unit” can be “anything” a person defines it as – as long as it’s defined – and recognize how this comes into play in the lab
• Know types of enzyme inhibition (competitive, uncompetitive, noncompetitive, mixed) – will be reinforced in lab
  • Be able to identify what effects they’d have on kinetic parameters
  • Be able to recognize evidence of them on plots – and be prepared to draw the effects they’d have on plots
  • Be able to give examples of different types of inhibitors

Monosaccharides 

• Refresh your knowledge from o-chem
  • Be familiar with Haworth perspective formulas (for rings) & Fisher diagrams (for linear sugars)
  • Be able to describe structures…
    • as aldoses or ketoses
    • by # of carbons (pentose, hexose, etc.)
    • by # of sides of ring structure (pyranoses, furanoses)
• Appreciate that there is a core set of monosaccharides, but these can also have derivatives (e.g. amino sugars, deoxy sugars, acidic sugars)

Disaccharides

• Know that monosaccharides are joined together through O-glycosydic bonds
• Know components of lactose (glucose & galactose) & sucrose (glucose & fructose)

Polysaccharides

• Appreciate storage vs. structural functions & what characteristics are ideal for what purposes 
• Compare & contrast glycogen, starch, cellulose, & chitin

Glycoconjugates 

• Know types of glycoconjugates (glycoproteins, proteoglycans, glycolipids, etc.)
• Be familiar with how glycosylation (enzymatic) and glycation (non-enzymatic) occur in vivo 
• Identify some roles glycoconjugates play in biology
• Be familiar with N- & O-linked glycosylation & identify where such glycation can occur (e.g. which amino acids can be N- or O-glycosylated)

Core concepts

• Define the terms “metabolism”, “anabolism”, “catabolism” and “anapleurotic” 
• Appreciate that metabolic pathways are really more like webs or interconnected subway systems
• Appreciate that some pathways are limited to particular tissues (commonly the liver) because the required enzymes are only expressed there
• Appreciate that different tissues and/or cellular compartments (e.g. mitochondria) use different versions of enzymes that may differ in their regulation and/or activity
• This stuff can get overwhelming if you just try to memorize it – instead, focus on:
  • Learning/reviewing fundamental underlying principles
  • Examining the starting and ending materials
    • Identify what needs to change
  • Walking through the pathway and tracking the flow of electrons
    • Be on the lookout for functional groups & cofactors
  • Understanding the purpose of each step
  • Recognizing which steps will be thermodynamically “easy” or “hard”
    • note that these steps may have alternative pathways in the reverse direction
  • Noting steps at which intermediates can join in or be siphoned off into other pathways
• Warning: different sources may use different abbreviations for the same molecule

Thermodynamics

• Review thermodynamics
• Reversible vs. “irreversible” reactions
  • Be able to explain the difference
  • Predict whether a given reaction will be reversible or irreversible
  • Explain the roles of irreversible reactions
    • commitment to a pathway, trapping of intermediates, etc.
• Explain how concentrations of products and reactants can be regulated to drive reactions in particular directions (review LeChatlier’s principle) 

Cofactors & coenzymes

• Know core coenzymes (organic enzyme cofactors) and what type of reactions they’re used for (learn them as you go along, but here’s a list of key ones to remember):
  • NAD(P)+, FAD: redox
    • you see these a lot in energy-generating pathways, as they act as electron carriers to take electrons to the ETC (electron transport chain)
  • PLP (pyridoxal phosphate): amino group transfer
    • will come into play a lot in amino acid metabolism
  • TPP/TDP (thiamine pyrophosphate): decarboxylation
  • Biotin: carboxylation
  • Tetrahydrofolate (THF) (from folic acid): one-C group transfer
    • seen a lot in anabolic pathways
  • Coenzyme-A (CoA): acyl group transfer
    • seen in acetylation reactions

Redox reactions (reduction & oxidation)

• Review redox principles
• Be able to Identify whether a reaction is a redox reaction
• Identify which molecules are being reduced and which are being oxidized in a given redox reaction
• Know cofactors often used in redox reactions
  • NADH, NADPH, FADH2
• Recognize how reactive oxygen species (ROS) can cause damage to biomolecules if not tightly controlled