CHEM 365 (Stindt) - Biochemistry I, Exam 2

Proteins and Enzymes

What types of chemical interactions dictate secondary structure?

Non-covalent interactions: IMFs, ionic bonds, disulfide bonds, hydrophobic interactions/hydrophobic effect

What are Phi and Psi?

Dihedral angles of polypeptides that determine secondary structure

What secondary structures do different Phi and Psi angles result in?

Alpha-helices and Beta-sheets (parallel and antiparallel)

What are the major features of alpha-helices? AAs, handedness…

Alanine included, Glycine and Proline not included, right-handed, net dipole along axis between N-terminus and C-terminus

Why are Glycine and Proline not included in alpha-helices?

Proline has a fixed shape, Glycine is too small and flexible

What types of amino acids interact with the electronegative carboxyl, C-terminus? Why?

Positively charged amino acids, the interaction is stabilizing

What types of amino acids interact with the electropositive N-terminus? Why?

Negatively charged amino acids, the interaction is stabilizing

Where does H-bonding occur on an alpha-helix?

Between the H attached to the electronegative nitrogen and the electronegative carbonyl O

What are the major features of beta-sheets? AAs, bonding…

Proline and Glycine included, antiparallel and parallel sheets, H-bonding occurs on adjacent segments of the polypeptide within the sheet

What is the difference between H-bonding on antiparallel vs parallel beta-sheets?

Antiparallel H-bonding occurs in straight lines, parallel H-bonding occurs out of lines on angles

parallel is in-line and antiparallel is out of line

What does a Ramachandran plot show?

Shows typical angles for Phi and Psi in secondary structures, show abundance of alpha-helices and beta-sheets, shows heat maps of all peptides in a protein against both angles

How does circular dichroism work/show?

Uses polarized light to show the abundance of left vs right handed peptide bonds

What amino acid is found in a Type I beta-turn?

Proline

What amino acid is found in a Type II beta-turn?

Glycine

What is the hydrophobic effect?

When waters around a hydrophobic thing order themselves to make fewer favorable reactions with other waters

What is the difference between globular and fibrous proteins?

Spherical, globular shape vs. long strands or sheets

What are the structures of fibrous proteins?

Repeated alpha-helices and repeated beta-sheets

Examples of fibrous proteins?

alpha-keratin (hair), fibroin protein (silk)

Examples of globular proteins?

Myoglobin, hemoglobin

What is a protein domain?

Distinct functional sections within a protein

What is a motif?

The arrangement of a secondary structure into a tertiary structure

What are some common motifs?

Beta-alpha-beta loops, beta-barrels

What is proteostasis?

A process that maintains the native structure of a protein

Role of a chaperone protein?

To help other proteins fold correctly into their native structures and help misfolded proteins fold back to their native structures

Role of a protease?

To get rid of misfolded proteins or proteins that are no longer needed

What is an amyloid fibril?

A beta-sheet aggregate that encourages other beta-sheets to bind to it

What is a prion?

A normal brain protein that contains beta-sheets that can form infectious aggregates

How does mass spec work?

A molecule is passed at a great speed along a charge gradient and a mass/charge ratio is read out

What information can a mass spec provide?

Highly accurate molecular weight, some can provide sequences of polypeptides

What is the difference between ESI and MALDI?

ESI vaporizes solutions by passing them through a needle at a high speed, MALDI blasts samples with lasers on a matrix (solution sample vs solid sample)

Similarities between ESI and MALDI?

They both measure mass and can give information on sample composition

What is X-Ray crystallography used for?

To get a diffraction pattern (structural data) on solid samples

What are the drawbacks of X-Ray crystallography?

Some samples are hard to crystallize, some limitations on structural information, can only show one conformation

What is Cryo-EM used for?

To get 3D images of the structure of liquid samples and can get multiple conformations

What are the drawbacks of Cryo-EM?

Requires heavy computation to find the structure and piece together images, can be lead by a hypothesis

Describe protein binding (using ligand, binding site, and induced fit)

Protein binding involves binding of a ligand to the protein’s binding site, induced fit is the "fit" of the ligand at the binding site

What is the association constant?

The affinity of the ligand for the protein

What does a low dissociation constant mean?

Reaction favors bound product, Kd = 1/Ka

What is Y?

Fractional occupancy, fraction of sites bound, Y = bound protein concentration/total protein concentration

What is cooperativity?

How a binding event affects subsequent bindings

What is an allosteric protein?

A protein in which the binding of the ligand to one site affects the binding properties on another site

What are the 3 types of cooperativity and what do they mean?

Negative: binding event makes subsequent bindings less likely, Positive: binding event makes subsequent bindings more likely, Non: binding event has no effect on subsequent bindings

How does nH (the Hill coefficient) relate to cooperativity?

nH < 1: negative cooperativity, nH = 1: non-cooperativity, 1 < nH < number of binding sites: positive cooperativity

What is an antigen?

A foreign agent

What is an epitope?

A specific chemical group (ligand) that antibodies bind to

What is variable domain?

The N-terminus of the heavy and light chain where the antigen binding site is

What is a polyclonal antibody?

A mixture of antibodies generated against a singular antigen, generated by multiple B cells, recognizes multiple epitopes

What is a monoclonal antibody?

An antibody that is generated by a single B cell, recognizes a single epitope

What are the different (common) immunoglobulin types?

IgG, IgA, IgE, IgM

What is an ELISA?

It is a way to screen for the existence of an antigen. It uses 2 antibodies; one to bind to the antigen of interest and one to report the presence of the antigen

What is a Western Blot?

Screens for the existence of a protein. It is done on an SDS-PAGE gel and separates proteins by size, then the two antibodies are applied (one to bind and one to report)

What is a substrate?

The enzyme version of a ligand

What is a cofactor?

A non-protein chemical component that coordinates with an enzyme to work

What is an organic cofactor called?

Coenzyme

What thermo property to enzymes affect?

They lower the transition state

How do enzymes lower transition states?

By having favorable reactions with substrates

Describe induced fit for an enzyme

When an enzyme undergoes conformational change when the substrate binds

What kind of interactions occur with induced fit of an enzyme?

Weak interactions between the substrate and enzyme

What is a ternary complex?

A non-covalent interaction between multiple substrates and an enzyme

What is enzyme ground state?

The starting point for either forward or reverse reactions in terms of either S or P

What step does a competitive inhibitor act on?

The binding of the substrate to the enzyme

What step does an uncompetitive inhibitor act on?

The conversion step (ES→EP)

What step does a mixed inhibitor act on?

Both the binding step and conversion step

What does a competitive inhibitor change?

Slope, Km

What does an uncompetitive inhibitor change?

Y-intercept, Vmax

What bonds are used in reversible inhibitors?

IMFs

What bonds are used in irreversible inhibitors?

Covalent bonds

What is a mechanism-based inhibitor?

Inhibitor that hijacks the normal enzyme reaction mechanism to inactivate the enzyme and covalently link to it

What is a transition-based inhibitor?

Inhibitor that binds to an enzymes transition state and covalently link to it

What is the use of mechanism-based inhibitors in pharmaceuticals?

Rational drug design

What is rational drug design?

A specific enzyme that is inactive until it reaches the activation site

What uses do transition-state inhibitors have in pharmaceuticals?

Protease inhibitors

Why don’t Michaelis-Menten kinetics apply to irreversible inhibitors?

They don’t apply because the kinetics rely on reversible, equilibrium reactions

What is enzyme activity?

The amount of conversion of substrate to product

What is enzyme activity based on?

The amount of enzyme at optimal conditions

What is specific activity?

Units of enzyme activity per total protein

What is an allosteric enzyme?

An enzyme that undergoes conformational change in response to binding with an allosteric modulator

What is an allosteric modulator?

Usually a small metabolite or cofactor that binds somewhere other than the active site, reversibly bound

What is an allosteric modulator that is a substrate?

A homotropic modulator

What is an allosteric modulator that is NOT a substrate?

A heterotropic modulator

How do allosteric modulators affect M-M kinetics?

They can change activity negatively or positively, change Vmax and/or Km, tend to show sigmoidal curves

What enzymes do the phosphorylation modifications?

Kinase adds a phosphoryl group and phosphatase removes it

What is a regulatory cascade?

A chain reaction of covalent modifications coming from a single stimulus

What type of enzymes are kinase and phosphatase?

Covalently-modified enzymes

Where do covalently-modified regulators bind?

Somewhere other than the active site