Biochemistry notes Mcat PDF

Preview

Summary

mcat notes...

Description

Biochemistry MCAT notes Chapter 1: amino acids, peptides and proteins 

 

Amino acids contain 2 functional groups: o Amino group o Carboxyl group α-carbon is a chiral center, and this makes most a.a optically active o Glycine is an exception and is the only achiral molecule A.a in eukaryotes are all L-amino acids and have an absolute S configuration o Cysteine, however, is the only a.a that is an L-amino acid but has an R absolute configuration Nonpolar, non aromatic side chains

 

 

Glycine is the smallest a.a in this group From smallest to largest for alkyl side chin length: o Alanine (1) o Valine (2) o Leucine (3) o Isoleucine (4) Methionine is an a.a that contains a sulfur group, and due to the sulfur group being attached to a methyl group, it remains relatively non polar Proline is an a.a that has a 5 membered ring. It is the only a.a that has its nitrogen being attached to the α carbon and alkyl side chain. The ring also makes it lose flexibility and limit where it appears in a protein. Aromatic side chains



From smallest to largest: o Phenylalanine o Tyrosine- same configuration as phenylalanine, except has an OH attached to the ring o Tryptophan- contains a double membered ring system Polar side chains

   

these 5 a.a are polar but are not aromatic serine and threonine are a.a that contain OH groups in their alkyl side chains (allows for Hbonding) Asparagine and glutamine are a.a that contain an amide group in their side chain Cysteine contains a thiol (-SH) group in its side chain and is more prone to oxidation due to the -SH group having a weaker bond than the OH group

Negatively charged side chains (acidic)   

There are two a.a that at physiological pH (7.4) are acidic in nature, which are aspartic acid and glutamic acid Aspartic acid and glutamic acid have the same configuration as asparagine and glutamine except instead of an amide group they contain an OH group Aspartate and glutamate are the anion forms (deprotonated OH) Positively charged side chains (basic)



There are 3 a.a in this category o Lysine- has a terminal primary amino group in its side chain o Arginine: contains three nitrogen atoms that delocalize the positive charge o Histidine: contains an aromatic ring side chain with nitrogen atoms in it. As the acidity increases the 2nd nitrogen can become protonated, giving the a.a a positive charge



Hydrophobic a.a: alanine, valine, leucine, isoleucine, phenylalanine (found interiorly in proteins away from water) Hydrophilic a.a: negative and positive side chains as well as asparagine and glutamine



1.2: acid base chemistr y of a.a      

A.a are amphoteric species, meaning they can accept or donate a proton pKa is the pH at which halve of the a.a is protonated and deprotonated ionizable groups tend to gain protons at low pH and lose protons at high pH environments a.a have 2 or more groups that can be deprotonated. pKa1 is for the carboxyl group (pka=2), pKa2 is for the amino group (pka=9-10) and pka3 is for the side chain zwitterion ions are a.a that have a positive and negative charged group (HA=A -) Titrations of a.a: at 0.5 equivalents of NaOH, the a.a exists in equal amounts between its fully protonated form and zwitterion form o When 1.0 equivalents have been added, the a.a exists as its zwitterion form only and the pH is equal to the isoelectric point of the a.a (molecule is neutral) o pI of a. a= (pkaNH group +pka COOH group)/2 o pI of a.a acidic= (pka R group +pka COOH group)/2 o pI of a.a basic= (pka R group +pka NH group)/2 o at 1.5 equivalents the zwitterion form and fully deprotonated form are of the a.a are equal o at 2.0 equivalents the a.a is fully deprotonated o in acidic a.a with side chains deprotonation occurs first at the carboxyl group, then the side chain and then the amino group o in basic a.a with side chains deprotonation occurs first at the carboxyl group, then the amino group and lastly the amino side chain group o acidic a.a have a +1 charge like other a.a when fully protonated and basic a.a have a +2 charge

1.3: peptide bond formation        

peptides are made up of a.a subunits called residues dipeptides are two a.a residues bonded together,etc 20 residues=oligopeptides, >20=polypeptides Peptide bonds are formed through the bonding of the COOH group and NH group of an a.a Peptide bond formation is considered a condensation/dehydration rxn, forming a water molecule as a product Due to the delocalizable π electrons a.a peptide bonds have double bond characteristic, causing the protein backbone around these bonds to be more rigid and have restricted rotation N-terminus is the amino end of a peptide and the c-terminus is the carboxyl end Peptide bond breakage occurs through hydrolysis. This is done by adding a H to the amide nitrogen and OH group to the carbonyl carbon

1.4: primary and secondary protein structure Primary structures are a linear arrangement of a.a Secondary, tertiary and quaternary structures are the most energetically favourable arrangements of the primary structures  Secondary structures assume to formations and are held together through hydrogen bonding o α-helices- rod like in shape and has the side chains pointing away from the core (core component of keratin) o β-pleated sheets: can be parallel or antiparallel to one another and are held together by hydrogen bonding o proline is rarely found in the center of the two formations due to the kinks it forms, however it is found in the beginning of a α-helices and turns in β-pleated sheets. 1.5: tertiary and quaternary protein structure  tertiary structures: is determined by hydrogen bonding, hydrophobic/hydrophilic interaction and acid-base interactions.  Salt bridges are formed between two amino acids with a charged R group (ex: cysteine) when the a.a are oxidized losing 2 hydrogens and electrons to form disulfide bonds  Disulfide bonds produce a loop in proteins and determine how curly a person’s hair is  The reason why hydrophilic residues are found on the surface of the protein and hydrophobic are in the interior is due to entropy. Hydrophilic residues increase entropy because they allow the water molecules more freedom in their positioning, which makes the solvation process spontaneous. Hydrophobic residues decrease entropy due to them causing water molecules to have less freedom(latitude) causing them to assume specific arrangements to maximize hydrogen bonding, which decreases entropy. This makes the solvation process nonspontaneous  Quaternary structure: only exists in proteins that contain more than one polypeptide chain and are an aggregate of smaller globular peptides and represents functional form of protein (ex: hemoglobin)  



 

Formation of quaternary structure has several roles o They can increase stability by decreasing surface area o They can reduce the required amount of DNA needed to encode the protein complex o Can bring enzymatic sites closer together enabling the swift shuttling of intermediates from one site to another o Can provide allosteric effects/cooperativity, which can enhance or inhibit the activity of other subunits when there is a conformational change of a particular subunit. Conjugated proteins are proteins that derive their functions from molecules called prosthetic groups. There are three groups commonly known: o Lipoprotein-lipid o Glycoprotein-carbohydrate o Nucleoprotein-nucleic acid

1.6: Denaturation of proteins   

Denaturation is an irreversible process where proteins are broken down through the use of heat or solutes. Heat messes with proteins by providing enough energy to overcome the hydrophobic interactions that hold the protein together, causing it to unfold. Solutes mess with the protein structure by directly interfering with the intramolecular forces or bonds that hold the protein together (ex: hydrogen bonding, disulfide bonds)

Chapter 2: Enzymes 

 

Key points about enzymes: o Lower activation energy o Increase rate of rxn o Are not changed/used in rxn o Heat/pH affect enzymes optimal activities o Do not affect the overall ∆G of the rxn o Are specific for certain rxns Enzyme specificity: an enzyme will only catalyze a single rxn/ class of rxns for a substrate There are six categories for enzymes: o Oxidoreductases: these enzymes carry out oxidation-reduction rxns. They transfer an efrom one molecule to another. The molecule that accepts the e - is a oxidant and the one that gives the e- is a reductant. o Transferases: catalyzes the movement of a functional group from one substrate to another. o Hydrolases: catalyzes the breakdown of a single molecule into two using water. o Lyases/synthases: does the same thing as hydrolase but without the use of water

Isomerases: catalyzes the rearrangement of bonds within a molecule (constitutional isomers or stereoisomers) o Ligases: catalyzes the addition/synthesis of two large molecules into one. Smaller molecules are done by lyases. Endergonic rxns require energy (∆G>0) while exergonic rxns give off energy (∆G> [S}: v= (kcat/km)[E][S] kcat/km is known as the catalytic efficiency of the enzyme Lineweaver-burk plots: a double reciprocal graph of the M-M equation

  

 

X-axis = -1/km, y-axis= 1/vmax The plot is useful for determining the type of inhibition the enzyme is experiencing Cooperativity: some enzymes can produce a sigmoidal graph instead of a hyperbola and this is due to the enzymes having multiple active sites and subunits. This provides the enzyme with two states (T state=low affinity tense state, R state= high affinity relaxed state). Substrates binding to the enzyme can encourage the transition from T state to R state and vise versa Cooperativity can be measured using hill’s coefficient: o If the coefficient is 1 then the enzyme is displaying positive cooperative binding (R state is induced) o If the coefficient is =1 then the enzyme is displaying no cooperative binding

2.4: effects of local conditions on enzyme activity 

Factors that effect enzyme activity: o Temperature: for every 10*C increase the velocity of a rxn tends to double until it reaches optimum temperature. After the opt temp, the activity falls off o pH: changes in pH can affect the ionization of active sites of an enzyme and can denature it as well o Salinity: increasing salt levels can disrupt hydrogen bonding and ionic bonds causing a partial change in the conformation of enzymes or denaturation

2.5: regulation of enzyme activity    

Feedback regulation: enzyme activity can often be controlled by products made further down the line of a metabolic pathway. Feed forward regulation: intermediates of a metabolic pathway proceding an enzyme may increase that enzymes activity Negative feed back: helps in maintaining homeostasis and involves the inhibition of the activity of an enzyme. Reversible inhibition: there are four types of reversible inhibitions: o Competitive inhibition: inhibitors and substrates compete for the active site of an enzyme. Inhibition can be overcome by increasing the substrate concentration. Adding inhibitors does not change the vmax because as long as enough substrates are added, the rxn will run at maximum velocity. It does however increase km due to it reducing the enzymes affinity for a substrate o Noncompetitive inhibition: this involves the inhibitor binding to an allosteric site of the enzyme causing it to not undergo a conformational change to allow binding to the substrate. This type of inhibition does decrease vmax because it decreases the number of available enzymes. Does not change km because the remaining active enzymes do not lose their affinity for their substrate. o Mixed inhibition: an inhibitor is able to bind to either the enzyme or enzyme substrate complex but has different affinities for each. This type of inhibition can affect km depending on the inhibitors preference ( enzyme binding= increased km=low affinity,

 

enzyme substrate complex= decreased km= high affinity). In either case vmax is still decreased as a result o Uncompetitive inhibition: the inhibitor binds only to a enzyme substrate complex. This in turn increases the affinity of the enzyme, but decreases the availability of it. This causes km to decrease and vmax as well. The plot for activity with or without the inhibition are parallel Irreversible inhibition: conformational change or activity site usage by the inhibitor is pretty much permanent making the enzyme completely unavailable. Regulated enzymes: o Allosteric enzymes: enzymes that contain multiple binding sites and usually these allosteric sites control the activity of the active site when bound to another molecule. They have an active/inactive form. Molecules binding to these sites are either allosteric activators/inhibitors. o Covalently modified enzymes: enzyme activity can be affected by the addition or removal of a prosthetic group, such as the dephosphorylation or phosphorylation of an enzyme. o Zymogens: some enzymes can be extremely dangerous if they are not controlled in the body. To avoid any damage, these types of enzymes are usually released in an inactive form. They also contain an active domain and regulatory domain. Regulatory domain must be removed in order for the active site to be available.

Chapter 3: nonenzymatic protein function and protein analysis 

  



Structural proteins: there are five types of structural proteins and they contain repetitive organization of 2nd structural elements, which is known as a motif o Collagen: is important in providing strength and flexibility to the body and makes up most of the extracellular matrix. o Elastin: another important part of the ECM which adds stretching and recoil to tissues. o Keratins: contributes to the mechanical integrity of the cell and functions as a regulatory protein (found in hair and nails) o Actin: protein that makes up microfilaments and has a positive and negative side that allows motor proteins to move unidirectionally in eukaryotic cells o Tubulin: protein that makes up microtubules and is important in structure, chromosome separation and intracellular transport Motor proteins: has transient interactions with actin and microtubules and can act as an ATPase to power a conformational change required for movement. Myosin: primary motor protein that interacts with actin. Is a thick filament in myofibril and can be involved in cellular transport Kinesins: plays a role in aligning chromosomes during metaphase and depolymerizing the microtubule during anaphase. Also transports materials towards the positive part of the microtubules. Dyneins: cause the sliding movement in flagella and cilia and are responsible for transporting materials towards the negative end of the cell (inwards)

 





Binding proteins: proteins that have stabilizing functions by sequestering or transporting molecules and binding to them (ie. Hemoglobin) Cell adhesion molecules (CAM): proteins on the surface of cells that enable the cell to bind to another protein or the ECM o Cadherins: a type of glycoprotein which mediates calcium dependent adhesion. Also they are usually used to adhere two cells that are the same and are specific to each cell o Integrins: protein containing two membrane spanning chains called α and β which are important in communicating and binding to the ECM. Also plays a role in cellular signalling and can impact cellular function (apoptosis, cell division, etc.) o Selectins: protein that binds to carbohydrates found on the surface of other cells nd plays a role in immune defense like integrins (white blood cell migration. It is also the weakest biding out of the CAMs Immunoglobulins/antibodies: proteins produced by b-cells that are used to neutralize targets (toxins and bacteria) as well as recruit other cells to eliminate the threat. Every antibody has an antigen binding region which contains a specific sequence, found on the tip of the Y, that allows for the antibody to bind to only one specific antigen. The rest of the antibody is known as the constant region, which is involved in recruiting and binding to other immune cells (ie microphages When antibodies bind to their targets (antigens) three things can happen: o The antibody can neutralize the target o The antibody can mark the target to be destroyed by other white blood cells (opsonization o The antibody can clump around the target and be phagocytized by a microphage

3.2: biosignaling   



 

Biosignaling is a process by which cells receive or act upon a signal Ion channels: proteins that provide a pathway for charged molecules to enter or leave the cell Facilitated diffusion: type of passive transport that involves the diffusion of substrates down a concentration gradient through a pore in the cell membrane provided by a transmembrane protein There are three types of channels: o Ungated channels: posses no gates and is not regulated (example: potassium channels) o Voltage gated channels: channel that is regulated by membrane potential change in the cell. o Ligand gated channels: channel that is controlled by the binding of a substance or ligand causing the channel to open or close. Km and vmax can also be applied to transporters Enzyme linked receptors have three main domains o Membrane spanning domain: anchors the receptor in the cell membrane o Lingand binding domain: where the ligand binds, inducing a conformational change and activating the catalytic domain o Catalytic domain: the area where another enzyme will be activated through different means such as phosphorylation and is usually the start of a 2 nd messenger cascade.







G-protein coupled receptors: integral membrane proteins involved in signal transduction. Each receptor is specific to a designated ligand. In order for a GPCR to transmit signals to an effector cell they use a heterotrimeric G protein. Binding of a G-protein causes it to enter its active form and affect the intracellular signalling pathway. There are 3 main types of G proteins: o Gs: stimulates the adenylate cyclase, which increases cAMP levels o Gi: inhibits adenylate cyclase, which decreases cAMP levels o Gq: activates phospholipase C, which increases calcium levels in the cell The three subunits that comprise g proteins are alpha,beta and gamma. o In its inactive form the alpha subunit is gdp and forms a complex with beta and gamma subunits. When a ligand binds to the GCPR, a receptor becomes activated and the corresponding g protein is activated. Once GDP is replaced with GTP, the alpha subunit dissociates from the complex and goes to adenylate cyclase and causes a change in its activity depending on whether it is an alpha-s or alpha-i subunit.once GTP is dephosphorylated back to GDP, the alpha subunit binds to the beta and gamma subunits again and the g protein becomes inactive

3.3: protein isolation   



         

Electrophoresis: involves subjugating proteins to an electric field causing them to move based on their charge and size Polyacrylamide gel: standard medium for protein electrophoresis Polyacrylamide gel electrophoresis (PAGE): used to analyze proteins in their native states by comparing similar sized proteins based on their molecular size and charge. This form is limited due to proteins varying mass to size and mass to charge ratios due to multiple different proteins exhibiting the same level of migration. Pros is that you can reuse the protein as long as it hasn’t been stained. SDS page: is used to separate proteins based on their molecular mass by using SDS, a detergent, which neutralizes the charge of the protein and causes the protein to be only affected by the electrical field and the frictional coefficient (which depends on mass) Is...