A measure of how much light is absorbed by a chromophore. Measured by a spectrophotometer.

I₀ = intensity of light going into the cuvette

I = intensity of light after the cuvette

A = log₁₀(I₀/I)

The molecule in a hydrogen bond which has an electronegative atom with unpaired electrons, such as O or N. A common acceptor is water.

Amino acids which are hydrogen acceptors:

D, E, H, N, Q, S, T, and Y

aka Aspirin

Inhibits the enzyme cyclooygenase, which catalyzes synthesis of prostaglandins which contribute to inflammatroy response. A natural compound, acetylated.

aka Energy of activation

aka Energy barrier

A factor in the Arrhenius equation. Always a positive number. The threshold energy required for a reaction. Enzymes decrease activation energy by chemical catalysis at a relatively low temperature and neutral pH.

[image]

An amino acid.

Very non-polar. Small. Forms α-helices.

Mass = 71.03712 Da

- C(NH₂)=O

A polar group.

A small molecule. An α-carbon with:

-a carboxylate group

-a hydrogen

-an R side chain

There are 20 different amino acids, which differ by their side chains.

Electrolytes. Mass approximately 110 Da. The building block of proteins. Forms amide bonds.

Necessary for determining protein structure.

Two steps:

1. Separation of the mixture into components.

2. Detection of components.

-NH₂

A component of an amino acid. Forms amide bonds with carboxylate groups of other amino acids.

Invented by Christian Anfinsen.

Denaturing of the protein ribonuclease using 2-mercaptoethanol and urea, reducing disulfide bonds and unfolding the protein. The protein can be refolded into its native state by first removing urea using dialysis and then exposing the protein to oxygen to reform disulfide bonds. The enzyme regains its enzymatic actvity. Skip one step, or do the steps in the wrong order, and enzymatic activity is not recovered.

This experiment proves that proteins fold the way they do based off of primary structure alone; not by a specific folding process.

1 x 10^-10 m

A unit of length used for measuring atomic structures.

The length of a C-H bond is one Å.

An amino acid.

[image]

Very polar. Positive charge. Basic. Forms α-helices. A hydrogen donor. A target of trypsin, except when followed by Pro.

pKa = 12.5

Mass = 156.10112 Da

Used to calculate the rate of reaction.

Rate = p Z e^-Ea/RT

A compound with structure sufficiently similar to a substrate so that enzyme binds to it and produces a product with a distinctive property. Used in enzyme assays.

Example: p-nitroaniline

An amino acid.

[image]

Very polar (due to amide group). Negative charge. Derived from aspartate. A hydrogen donor and acceptor. A secondary structure breaker: it forms H-bonds with the adjacent backbone, disrupting the H-bonds of secondary structure. 

Mass = 114.04293 Da

An amino acid.

[image]

Negative charge. The "brother" of glutamate. When protonated, it is called aspartic acid. A secondary structure breaker; forms H-bonds with the adjacent backbackbone, disrupting secondary structure H-bonds. A hydrogen acceptor.

pKa = 4.0

Mass = 115.02695 Da

A combination of Beer's Law and Lambert's Law.

A = ε c l

Basic Local Alignment Search Tool

Compares input sequences to all databanks of all known protein sequences and produces the best match. A 100% identical finding would be a positive match. Good for identifying homologs and new proteins. Biochemical tests used to determine identities. Only 40 - 50 amino acids need to be sequenced to identify a protein.

A substance present in solution in sufficiently high concentration to control the pH. Creates a mixture of protonated and deprotonated forms, so that the mixture is at equilibrium near the pKa of the buffer. 

Example: bicarbonate ions, phosphate ions, phosphate esters, dihydrogen phosphate, and Tris.

The end of a polypeptide ending with a carboxyl group. The last amino acid listed. Represented by the red end of the ribbon in a ribbon model. Negative charge.

pKa = 2.4 or 3.

One of the elements found in the body.

Charge = +2

Atomic number = 20

Atomic weight = 40.08

One of the elements found in the body.

Charge = -4, +2, or +4

Atomic number = 6

Atomic weight = 12.09

-C=O

The point of weakness in an amide bond.

-COO^-

A component of an amino acid. Forms amid ebonds with amino grouops of other amino acids. The deprotonated form of carboxylic acid. Very polar and negative.

-COOH

A proton donor. The protonated form of carboxylate.

The catalytic unit of chymotrypsin.

Aspartate 102, Histidine 57, and Serine 195

They are close together in the enzyme due to folding.

A protease that severs polypeptides at F, W, and Y, except when followed by P. All fragments will have a F, W, or Y residue on the C-terminus, except for the C-terminus fragment. Has a large binding pockets lined with G, L, and W, designed for large, non-polar amino acids. Forms H-bonds with the backbone C=O and NH groups, positioning the polypeptide so that the catalytic triad oxyanion hole severs the correct peptide bond. Reacts 40 times a second. Speeds up hydrolysis 4 x 10⁹ times.

The reaction is broken into two steps:

1. Nucleophile attacks the peptide bond, splitting the peptide chain and releasing the C-terminus, forming an acyl-enzyme intermediate.

2. Water is used, releasing the N-terminus and returning the enzyme to its original state.

A type of amino acid separation. The stationary phase is a resin and the mobile phase is a buffer, placed in a column. Compounds allowed to flow through thhe column and are separated by their characteristic elution volume. Particles more attracted to the stationary phase require higher elution volumes. Samples of the separated substances can be collected for further investigation.

Includes: high performance liquid chromatography, ion exchange chromotography, metal affinity chromatography, and gel flitration.

An inhibition mechanism where the inhibitor can only bind to an enzyme that is not bound to substrate. Has the same shape and properties as the substrate and binds to the same active site. Substrate and inhibitor compete to occupy the enzyme. V_max is unchanged, but K_M increases. The slope of the Lineweaver-Burk plot increases while the Y-axis stays the same.

K_i = [E][I]/[EI]

K_M'=K_M(1 + [I]K_i)

States of a molecule that can be interconverted by breaking covalent bonds.

Example: cis and trans configurations of a C=C double bond, and chiral forms of amino acids.

The first step in Edman degradation. Occurs in a high pH of 9 (basic), so phenylisothiocyanate is attacked by the nucleophilic, deprotonated N-terminus. They bond, forming phenylthiocharbamoyl peptide.

The reaction must be complete before cyclization can take place.

CNBr

Cuts polypeptides at M residues (regardless of presence of P). Attacks the S atom of methionine. Peptide backbone is broken at on the C-terminus side of methionine. Methionine is converted into homoserine. All fragments have a homoserine on the C-terminus end, except for the C-terminus fragment.

The second step in Edman degradation.

Occurs in high pH (acid). Weak anhydrous acid attacks the C=S bond in phenylisothiocyanite, causing he S atom to react with the point of weakness in the amide bond, breaking off the N-terminal amino acid, producing a PTH amino acid.

Not a hydrolysis reaction; no water is present. The reaction must bec complete before the next coupling can take place.

An amino acid.

[image]

Moderately non-polar (slightly polar SH group on β-carbon). No charge. Forms β-strand (large S atom on β-carbon). Forms disulfide bonds.

pKa = 8.5

To lose a proton. pKA tells what pH half of molecules are deprotonated. If the pH is one higher than pKa, it can be assumed that all molecules are deprotonated. The deprotonated charge is one less than the protonated charge.

If the atom is O or S, charge is -1

If the atom is N, charge is 0

H₂PO₄^-

A buffer used in labs. Keeps pH near 6.8.

aka Folding units

Sectioins of proteins. Large proteins (50+ kDa) may have many domains. Small proteins may have just one. Domains are around 10 - 20 kDa in size. Folds as an independent entity. Domains may belong to differing tertiary structure family.

The molecule in a hydrogen ond which has a highly polar -OH or -NH group. A common donor is water.

Amino acid donors:

H, L, N, Q, R, S, T, and Y

Tendency to hold bonding electrons.

O > N > S > C ≈ H

Atoms with similar electronegativity have non-polar bonds. Atoms with more different electronegativity has polar bonds.

"Electron-loving"

Atoms with a positive charge that attract nucleophiles. May be subject to nucleophilic displacement reactions. There are no very good electrophile amino acids.

(Rate of reaction) x (reaction volume)

Moles of substrate converted per unit of time.

A measure of the quantity of enzyme present. SI unit is the katal, but also measured in enzyme units.

100% x (specific acitivty of enzyme sample / specific activity of pure enzyme)

A measure of the purity of an enzyme sample. No sample is completely pure.

1 μmol/minute

A unit of enzyme activity.

aka β-strand

A secondary protein structure. Every α-carbon down the peptide chain turns 109º in alternating directions. The distance between adjacent amino acids is 3.5 Å and the distance betwen amino acids facing the same side is 7.0 Å. Represented by an arrow pointed from the N-terminus to the C-terminus. Forms hydrogen bonds with other β-strands, forming a β-sheet. The amino acids C, F, I, T, V, W, and Y prefer to form β-strands because they are large or have a branch or group on the β-carbon. 

aka β-keratin

A protein found in moth and spider silk. A continuous antiparallel β-strand.

rate = k[S]¹

Rate plotted vs. [S] makes a straight line with a slope.

An amino acid.

[image]

Very polar. Negative charge. The "brother" of aspartate. Forms α-helices. A hydrogen acceptor. When protonated it is glutamic acid.

pKa = 5.0

An amino acid.

[image]

Polar (amide group). Uncharged. Derived from glutamate. A hydrogen donor and acceptor. Forms α-helices.

An amino acid.

[image]

Moderately non-polar. Hydrophobic. The smallest amino acid. The only amino acid with a non-chiral α-carbon. A secondary structure breaker (too flexible; too small)

Mass = 57.02147 Da

aka α-helix

Secondary protein structure. Every α-carbon down the peptide chains turn 109º in the same direction. Rotates fully once every 3.6 amino acids. The helix extends 5.4 Å per turn and 1.5 Å per amino acid. Always a right-handed helix. Hydrogen bonds form between the C=O group and the NH group of the amino acid five amino acids down the backbone. The inside of the helix is perfectly packed with atoms; there is no "hole". Represented as a curled ribbon in ribbon molecules. Side chains hang off the outside of the helix.
Amino acids A, E, F, H, K, L, M, Q, and R prefer α-helices because their side chains protect the backbone H bonds from disruption by water. 

Relates pH, pKa, and the state of ionization.

pH = pKa + log([deprotonated]/[protonated])

An important amino acid.

[image]

Very polar. Positive charge. Basic. Forms α-helices. Hydrogen donor and acceptor.

Can form his-tags for metal affinity chromatography.

At pH 7, 76% of histidine residues are protonated, giving it a +0.24 charge.

pKa = 6.5

 One of the elements found in the body.

Charge: -1 or +1

Atomic number: 1

Atomic weight: 1.008

"Water-loving"

Polar molecules that dissolve in water.

"Water-fearing"

Non-polar molecules that do not dissolve in water. Related to the number of CH, CH₂, and CH₃ groups present.

A molecule that forms covalent bonds with an enzyme, making it unable to complete its reaction. Usually irreversible. Reduces enzyme in a stoichiometeric manner.

Example: nerve gas.

1 + ([I] / K_i)

Appears in all inhibition equations.

An amino acid.

[image]

Very non-polar. Forms β-strands (branch on β-carbon). Has the same molecular mass as leucine.

1 mols

The SI unit for enzyme activity.

aka Fibrous protein

The simplest tertiary structure. One continuous secondary structure. Rigid and fibrous. No secondary structure breakers. Includes α-keratin, fibroin, and collagen.

1000 Da

The unit that proteins are measured in. The mass of 1000 H atoms. One Dalton (Da) is 1 g/ol.

A protein with two domains, both with a parallel αβ sandwich structure. Oxidizes the secondary alcohol in lactate, producing pyruvate. Rate of reaction is measured by the level of NADH produced, indicated by absorbance.

LD + NAD⁺ → pyruvate + NAD + H⁺

An amino acid.

[image]

Very non-polar. Forms α-helices. Has the same molecular mass as isoleucine.

A linear transformation. The reciprocals of both sides of the Michaelis-Menten equation. Makes a straight line.

1/v₀ = (K_M + [S]) / (V_max [S])

Y axis: 1 / v₀

X axis: 1 / [S]

Slope = K_M / V_max

Y-intercept = 1 / V_max

X-intercept (theoretical) = -1 / K_M

1901 - 1994

Won the Nobel Prize in Chemistry in 1954. Won the Nobel Peace Prize in 1962. Discovered the molecular structure and bonding of the α-helix and β-sheet. Built accurate scaled models with correct bond lengths, angles, and atomic radii. Found that peptide bonds have characteristics of double bonds; they are rigid and locked into tetrahedral sheets that limit polypeptides to only cis and trans configurations. This rigidity limits proteins into a stable native state. Found that there were only two types of protein structures: helical and extended. There are also random coils.

An amino acid.

[image]

Very polar. Positive charge. Basic. Target of trypsin, except when followed by P. Forms α-helices.

pKa = 10.2

Mass = 128.09497 Da

Matrix-Adsorption Laser Desorption Ionization-Time of Flight mass spectrometery

Mass spectrometry using a laser. Pure protein sample is vaporized by a laser, producing positively charged protein ion particles which drift towards a negative electrode and go through a small hole, forming a beam. The velocity of the particles is proportional to the ratio of mass to charge. Since charge is known, the mass can be calculated from the time of flight. The mass can be looked up in a BLAST search to identify it.

An amino acid.

[image]

Very non-polar. Forms α-helices. A target of cyanogen bromide.

Initiates all protein translations, but may be removed in post-translational processing.

(k₂ + k_-1) / k₁

The concentration of substrate that gives a rate exactly 0.5 V_max. A factor in the Michaelis-Menten equation. Typical values are betwen 10⁶ M and 10² M. Indicates the affinity between enzyme and substrate: a low K_M indicates a high affinity, and a low K_M indicates a low affinity.

Derived and tested in 1913 by Michaelis and Menten. A correlation between v₀ and [S]. When plotted it makes a hyperbolic line.

v₀ = (V_max [S]) / (K_M + [S])

or

(v₀ / V_max) = [S] / (K_M + [S])

The end of a polypeptide with an amino group. By convention, the first amino acid listed. Represented by the blue end of the ribbon in a ribbon model.

pKa = 9.6 or 8

National Centre for Biotechnology Information

A search engine with protein sequences, DNA sequences, RNA sequences, genomes, health-related mutations, PubMed, and more.

www.ncbi.nlm.nih.gov

Disopropylfluorophosphate

A highly toxic inactivator that inactivates acetylcholinesterase irreversibly, blocking transmission of nerve impulses.

One of the elements found in the body. When protonated it is positive. When deprotonated it is neutral.

Charge: -3 or +5

Atomic number = 7

Atomic weight = 14.09

An inhibition mechanism where the inhibitor can bind to the enzyme whether there is substrate bound to it or not. Binds to a different active site. Affects the catalysis mechanism of the enzyme. Inhibitor can bind to E, ES, but not EIS. V_max is lowered (if true non-competitive, it is halved). K_M stays constant. The slope of the Lineweaver-Burk plot increases while the X-axis stays the same.

V'_max = V_max / (1 + ([I] / K_i))

"Nucleus-loving"

An atom that has a lone pair of electrons available for sharing. Attracts positively charged electrophiles that they can share their electrons with. When it shares electrons, it forms bonds. Can act as a hydrogen bond acceptor, attracting OH or NH dipoles. Can act as a base and seek out H⁺ to be protonated. Can react in a nucleophilic displacement.

A small molecule that is the building block of nucleic acids. 1. a base

2. a sugar

3. a phosphate

Includes guanine, cytosine, thymine, and adenine.

One of the elements found int the body. When protonated it is neutral. When deprotonated it is negative.

Charge: -2

Atomic number = 8

Atomic weight = 16.00

aka TIM barrel

Named after triose phosphate isomerase, which has this structure. A very stable and common structure in nature. Many enzymes in glycolysis have this structure. Contains βαβ units. The α-helices all run parallel to each other and the β-strands all run anti-parallel to the α-helices (parallel to each other). β-sheets are mostly non-polar, so they form a β-barrel in the centre. Non-polar sides of α-helices attach to the β-barrel on the outside.

A structure that contains βαβ units. Polar α-helices are arranged on both sides of a polar parallel β-sheet, protecting it from contact with water. Usually the β-sheet is twisted, but does not form a full cylinder as in parallel αβ barrel structures.

Example: lactate dehydrogenase

An expression of the availability of H⁺ ions.

pH = -log₁₀[H⁺]

At pH = 7.0, [H⁺] = 10^-7 M

An amino acid.

[image]

Very non-polar. Large. Forms α-helices and β-strands. A target of chymotrypsin except when followed by proline.

Mass = 47.06842 Da

One of the elements found in the body.

Charge: -3, +3, or +5

Atomic number = 15

Atomic weight = 30.97

An amino acid.

[image]

Moderately non-polar. The R group links back around to the α-carbon, forming a 5-sided ring that makes proline rigid and inflexible. Secondary structure breaker (too rigid; disrupts H-bonding in backbone). The only amino acid which does not turn purple when exposed to ninhydrin; it turns yellow. It prevents chymotrypsin and trypsin from working if it follows the amino acid these proteases act on.

To gain a proton. pKa tells what pH half of a substance is protonated. When pH is 1 less than the pKa, it can be assumed that all molecules are protonated. Protonated molecules have a charge one greater than their deprotonated forms.

If the ionized atom is O, or S, charge is 0.

If the ionized atom is N, charge is +1.

The mobility of amino acids in thin layer chromatogrphy. Based on polarity: polar molecules have a lower R_F than non-polar molecules. Values are between 0 and 1. Amino acids are sorted and itentified by R_F.

X_b = how far up the amino acids have travelled.

X_f = solvent front

R_F = X_b / X_f

aka Ion pairs

A type of polar interaction. A strongly negative side chain has an electrostatic interaction with a strongly positive side chain nearby in the folded protein. Contributes to tertiary structure.

rate = k[S]²

When rate is plotted vs. [S], it makes an exponentially increasing line.

Glycine, proline, asparagine, aspartate, and serine.

A factor in secondary and tertiary stucture. When there are two or more together within a four amino acid window, they break α-helices and β-strands, maing a loop or turn section.

An amino acid.

[image]

Polar (OH group). No charge. Hydrogen donor and acceptor. The "brother" of threonine. A target of phosphorylation. A secondary structure breaker (forms H-bonds with adjacent backbone, disrupting secondary structure H-bonds).

Mass = 87.03203 Da

The rate of formation of enzyme-substrate complexes is equal to the rate of breakdown.

k₁[E][S] = k₂[ES] + k₁[ES]

One of the elements found in the body.

Charge: -2, +4, or +6

Atomic number = 16

Atomic weight = 32.07

aka MS/MS

A type of mass spectrmetry that diretly sequences proteins. Only small amounts of protein are required. Two-dimensional electrophoresis gels can be cut out and sequenced. The sample is selectively hydrolyzed and injected into a tandem MS. Two mass spectrometers in series: MS-1, collision cell, and MS-2. The sequence can be determined from the progression of mass that is produced.

An amino acid.

[image]

Polar (has OH group). No charge. Forms β-strands (branched β-carbon). Hydrogen donor and acceptor. The "brother" of serine. A target of phosphorylation.

Mass = 101.04768 Da

Trishydroxymethylaminomethane

A buffer used in labs. Keeps pH near 8.08.

An amino acid.

[image]

Moderately non-polar (very non-polar benzene ring, but also a NH group which slightly polarizes it). Forms β-strands (large size). Target of chymotypsin except when followed by P. The largest amino acid. Fluorescent.

Mass = 186.079322 Da

aka Molar activity

(specific activity) * (molar mass of enzyme)

Moles of substrate converted per unit of time per mole of enzyme.

An amino acid.

[image]

Moderately non-polar (very non-polar benzene, but the OH group makes it slightly polar). No charge. Target of phosphorylation. Forms β-strands (large). Hydrogen donor and acceptor. Target of chymotrypsin except when followed by P.

pKa = 10.0

Mass = 163.06333 Da

NH₂CONH₂

Used in the Anfinsen experiment. Unfolds proteins. Often used at a concentration of 8 M.

An amino acid.

[image]

A very non-polar amino acid. Forms β-strands (branched β-carbon).

Mass = 99.068842 Da

aka London dispersion forces

Weak eletrostatic attraction between neighboring atoms. Strength is 0.1 to 1.0 kJ. Strongly repulsive if atoms are too close, and fades if atoms are too far apart. Holds C atoms at approximately 3.4 Å distance. Contributes to protein tertiary structure. Maximizes close atom-to-atom contact like a jigsaw puzzle. Allows for enzymes to bind to substrates with complementary shape.

The theoretical upper limit of the observed rate of an enyme assay, when the enzyme is 100% saturated. A factor in the Michaelis-Menten equation. A higher V_max indicates a faster reaction. Proportional to the amount of enzyme used in the assay.

V_max = k[E_tot]

rate = k[S]⁰

Plotting rate vs. [S] produces a flat line.