Analytical Final - BCHM 322

rate

the speed at which a chemical reaction proceeds

concentration

chemical reaction rates depend on ________ of reactants

G = H - TS

what is the Gibbs Free Energy equation?

negative

In a chemical reaction, if the difference in free energy between the reactants and products is (positive/negative), the reaction is considered spontaneous

False

T/F: both directions of a reaction can be spontaneous.

• intrinsic properties of reaction itself (H, S)

• concentrations of products/reactants

what two things determines which reaction direction is spontaneous?

equilibrium

reactions will always proceed to the ________ state where the concentrations of reactants and products give a G=0

equilibrium

forward & reverse reactions are proceeding at equivalent rates and there is no net change in the concentrations of reactants and products

activation energy

needed to reach the higher energy transition state

catalyst

accelerates the rate of a chemical reaction; act by lowering the activation energy barrier of chemical reactions

True

T/F: catalysts are not consumed in a reaction

enzymes

catalysts produced by biological systems

substrates

reactants in enzyme-catalyzed reactions

• cell division

• energy generation

• neural function

• immunity from pathogens

enzymes are responsible for (4 examples):

• neutral pH

• atmospheric pressure

• ambient temperatures

enzyme reaction conditions (consider 3 things)

active sites

where the chemical reaction occurs in an enzyme; recognize and bind specific substrate structures among thousands of biological molecules in a cell

False

T/F: enzyme activity is always on

• binding affinity (uses optimal orientation)

• active site has appropriate chemical environment (charge, donors/acceptors)

• conformational changes

what are 3 ways enzymes increase reaction rates?

• energy conservation

• avoiding accumulation of toxic products

why must enzymes be regulated in biological systems?

• enzyme availability

• enzyme activation state

what are 2 general ways in which enzyme activities are controlled?

• feedback

• feed-forward

what are the two types of regulation?

feedback inhibition

one or more products of a pathway act as inhibitors of early steps in the pathway

feed-forward regulation

allows downstream steps in a metabolic pathway to be accelerated when intermediates begin to accumulate earlier in the pathway

• oxidoreductases

• transferases

• hydrolases

• lyases

• isomerases

• ligases

what are the 6 classes of enzymes?

oxidorecutactes

enzyme that transfers electrons to or from the substrate

transferases

enzyme that moves chemical groups from one molecule to another, breaking and forming bonds)

hydrolases

use water to break single chemical bonds in substrates

lyases

break bonds to eliminate chemical groups without hydrolysis or oxidation, generally resulting in the formation of a double bond or a new ring structure

isomerases

rearrange chemical bonds in molecules without changing the overall elemental composition

ligases

catalyze bond formation to join two substrate molecules/groups

True

T/F: enzyme reaction rates are a composite of the rates of individual steps

steady-state

an equilibrium condition in which the rate of ES formation is equal to ES dissociation + conversion of ES to E+P; the concentration of ES remains constant

>>

the steady-state is easily achieved when [S] __ [E] and lasts until [S] begins to be significantly depleted

enzyme assay

method to measure the progress of an enzyme-catalyzed reaction

• measure the disappearance of substrate over time

• measure the appearance of product over time

what are the two options for steady-state kinetic assays?

• spectral methods

• radioactivity

• electrophoretic imaging

common methods for monitoring reaction progress (3 things)

• linear response

• dynamic range

• specificity

• sensitivity

what are the four requirements for a useful enzyme assay?

continuous assay

measure product formation in real time

discontinuous assay

measure product formation after the reaction has been stopped

hydrodyamics

refers to the motion of particles in solution

sedimentation

movement of particles when a centrifugal force is applied to them

• mass

• shape

sedimentation behavior is dependent of a particles:

• centrifugal force

• friction

the velocity at which a particle sediments is dependent on two opposing forces:

centrifugal force

dependent on the particle’s mass, the speed at which the particle is spinning, and the distance of the particle from the center of rotation

friction

the frictional coefficient is determined by the size and shape of the particle and the viscosity of the medium it is moving in; dependent on the velocity at which the particle is sedimenting

sedimentation velocity

particles sediment at a constant rate

• pelleting

• fractional centrifugation

• density gradient centrifugation

what are three preparative methods for centrifugation?

• measuring apparent mass

• determining oligomeric state

• measuring binding affinities of protein-protein interactions

what are three analytical methods for centrifugation?

pelleting

generating soluble protein extracts from lysed cells or homogenized tissue

fractional centrifugation

isolation of specific cellular organelles by pelleting at incrementally increasing rotor speed

density gradient centrifugation

isolation of proteins, nucleic acids, or cellular organelles based on differences in size or density

effective mass

the centrifugal force felt by a particle during centrifugation is dependent on its:

sedimentation coefficient

relates sedimentation velocity relative to applied centrifugal force

• determining homogeneity of a sample

• measuring molecular weight

• determining oligomeric state

3 uses for sedimentation velocity centrifugation

• sample is mixed homogenously with solven and spun

• boundary is created w/ a particle-free area at the top of the sedimenting particles

• rate of movement of boundary is optically measured and provides sedimentation coefficient

• the width of the boundary zone provides diffusional coefficient

how is sedimentation velocity centrifugation done?

• Accurate measurement of molecular weight

• Determination of oligomeric state

• Measurement of dissociation constants for protein-protein interactions

what is sedimentation equilibrium centrifugation used for?

• A sample is spun at relatively low speed under physiological conditions until the frictional force = centrifugal force and molecules stop sedimenting (equilibrium)

• At this point, there is a balance between diffusion and sedimentation of sample molecules which creates a concentration gradient in the tube.

• The concentration from one end of the tube to the other is measured spectrophotometrically by the optical system of the ultracentrifuge (see figure).

• Importantly, the concentration gradient is dependent solely on the mass of the molecules and independent of shape. This allows for very accurate measurements of molecular mass.

• The analysis also reveals if other forms of the molecules are present (e.g. oligomers, aggregates)

how is sedimentation equilibrium centrifugation

• need a set of standards with known S values

• analyze the unknown sample in same way

zonal density gradient method

density; size

isopycnic separates particles based on _______; rate zonal separates particles based on ________

• protein purification

• sample desalting

• determination of molecular mass, protein shape

3 uses of size exclusion chromatography

• tolerant to different solution conditions

• elution is isocratic

advantages of size exclusion chromatography (2 things)

• beads must be inert

• beads must be chemically stable

• high concentration and small load volume of sample

what are the requirements for size exclusion chromatography?

stokes radius

influenced by a protein’s shape; hydrodynamic radius

enzyme kinetics

study of chemical reaction rates catalyzed by enzymes under different conditions

hyperbola

the Michaelis-Menten function is a _______.

Vo and [S]

Michaelis-Menten relates ___ and ____.

enzyme saturation

why does reaction velocity plateau at high [S]?

kcat

represents the number of catalytic events one enzyme molecule can perform each second

Km

reflects the affinity of enzyme for substrate; substrate concentration at ½ Vmax

• [E]<<[S]

• consumption of S must be minimal

• the initial rate must be measured

• the range of [S] must go above and below the Km

conditions for Michaelis-Menten kinetics (4 things)

• establish optimal reaction conditions

• perform control reactions

• estimate Km

• establish final conditions for determining Vmax and Km

steps for setting up Michaelis-Menton kinetics

Lineweaver-Burk plot

converts the Michaelis-Menton equation into a linear form by taking the reciprocal of both sides

curve fitting

more accurate method than the double reciprocal method of Lineweaver and Burk for determining the kinetic parameters

• hydrogen bonds

• Van der Waals forces

• Hydrophobic interactions

• Ionic bonds

the 4 types of noncovalent bonds that contribute to protein-ligand interactions

hydrogen bond

interaction between a hydrogen atom covalently bound to an electronegative atom and a second electronegative atom

Van der Waals forces

very weak interactions between molecular dipoles

hydrophobic interactions

interactions between two non-polar chemical groups that occurs in default in an aqueous environment

ionic bonds

interactions between chemical groups with negative and positive charges

True

T/F: a collection of noncovalent interactions are needed to establish physiological binding

the presence and arrangement of complementary chemical groups on the ligand and protein surfaces

what establishes specificity?

lock and key model

enzyme and substrate are rigid structures with fixed conformations

induced fit

substrate conformation is changed to stabilize transition state and allow chemical reaction

affinity & specificity

enzymes select substrates based on the principles of molecular recognition

catalytic efficiency

single value representing how efficiently an enzyme acts on a substrate

kcat/Km

catalytic efficiency is defined as:

Kd

dissociation constant; defined as [L][P]/[L-P]; half the binding sites on protein are occupied

equilibrium dialysis

two chambers separated by a semi-permeable dialysis membrane through which ligand, but not protein can pass freely

filter binding

proteins bind tightly to nitrocellulose filter discs, but most ligands won’t; mixture of protein and ligand are applied to a filter under vacuum, and protein and any bound ligand will be retained on filter

fluorescence

binding of protein to ligand will often affect the ___________ signal of a fluorophore on one of the molecules

• affinity purification coupled with mass spec

• yeast two-hybrid system

two methods for characterization of complex components

• co-immunoprecipitation

• fluorescence methods (FRET, Bimolecular fluorescence complementation)

• spectral methods (biolayer interferometry, surface plasmon resonance, dynamic light scattering)

methods for testing for interaction between specific protein of interest

yeast two-hybrid system

interaction of two proteins brings together the separated domains of a transcriptional activator that turns on expression of reporter gene that can be measured

FRET

occurs when the emitted light from one fluorophore excites a second fluorophore to emit light at a second wavelength; will occur when the two fluorophores are in close proximity to each other

co-immunoprecipitation

a protein of interest is purified out of a soluble protein extract from a biological source; the presence of co-purifying proteins are then monitored, typically by western blotting

biolayer interfermetry

protein is immobilized on the surface of a biosensor and light is transmitted through the sensor and the protein surface. Some of the light is reflected back from the protein surface and from the biosensor surface. A second molecule binding to the immobilized protein increases the thickness of the protein layer and changes the behavior of reflected light to that reflected by the biosensor surface. The change in reflected light behavior is measured and plotted.

competitive inhibition

inhibitor binds to the enzyme active site and blocks substrate binding; compete with substrate

increase; no change

competitive inhibitors (increase/decrease/no change) Km, and (increase/decrease/no change) Vmax

noncompetitive inhibition

bind enzymes at sites distinct from active site; allosteric inhibitor

no change, decreases

noncompetitive inhibitors (increase/decrease/no change) Km, and (increase/decrease/no change) Vmax

mixed inhibition

allosteric inhibition in which both Km and Vmax are effected