Biochem Flashcards
How do enzymes change a reaction?
Lowers activation energy and are not changed or consumed during the course of the reaction.
DeltaG remains unchanged.
What do dehydrogenases do?
Catalyze oxidation reduction reactions, not transfer reactions.
What does the Michaelis Menten equation tell us?
Enzymes form enzyme substrate complexes which can either dissociate back into the enzyme and substrate or proceed to form a product
What happens at high concentrations of substrate when the enzyme concentration stays the same?
Reaction rate approaches minimal velocity and is no longer changed by further increases in substrate concentration. This levels off the reaction rate after an initial increase.
What is a holoenzyme?
Biochemically active compound formed by the combination of an enzyme with a coenzyme
What is an apoenzyme?
Enzyme devoid of it’s necessary cofactor and is catalytically inactive
What is a coenzyme?
Non protein compound that is necessary for the functioning of an enzyme
What is a zymoenzyme?
Inactive precursor of an enzyme
What determines an enzymes specificity?
The three dimensional shape of the active site
What is Km?
This is the Michaelis menten constant.
Concentration of substrate which permits the enzyme to achieve half vmax.
What is Vmax?
Reaction rate when enzyme is fully saturated by substrate. All binding sites are being constantly refilled.
Example: vmax is near 100mmol/sec, vmax/2 equals 50mmol/sec. so the substrate concentration giving this rate is 0.5 mM and corresponds to Km.
This is from a chart
At high concentration values of substrate how if the rate of reaction affected?
It will be very close to Vmax if the concentration value is significantly larger than the value of Km.
Competitive inhibitors
Same Vmax and higher Km
Competes for active site
Increasing substrate can overcome this
Creates a X in the Burke plot
Noncompetitive inhibitors
Decreased Vmax and same Km
Binds to free E or ES complex
Increasing substrate can not overcome this
Doesn’t bind at active site
Km unaffected
Vmax reduced
Comes to a point for the Burke plot
Uncompetitive inhibition
Binds to ES complex only. Increasing the substrate favors the inhibition because it creates more ES complexes for it to bind to
Km reduced
Vmax reduced
Parallel lines
What is a lyase?
Responsible for breakdown of a single molecule into two molecules without the addition of water or the transfer of electrons
Often form cyclic compounds or double bonds in the products to accommodate this
What is a ligase?
Enzyme that catalyzes the joining of two large molecules by forming a new chemical bond. Usually with accompanying hydrolysis.
What is a hydrolase?
Use water to break a chemical bond which typically results in dividing a larger molecule to smaller molecules
What is a transferase enzyme?
Class of enzymes that enact the transfer of specific functional groups from one molecule (called the donor) to another (called the acceptor)
Cooperative enzyme
Demonstrates a change in affinity for the substrate depending on how many substrate molecules are bound. 3 substrates bound means higher affinity than 2 or 1 substrates bound.
Example is hemoglobin.
How does the idea temperature for a reaction change with and without an enzyme catalyst?
The rate of reaction generally increases with temperature because of the increased kinetic energy of the reactants but reaches a peak temp because the enzyme denatures. In the absence of an enzyme this peak temperature is generally much hotter.
What is the function of SDS in an SDS page?
SDS solubilizes proteins to give them uniformly negative charges so the separation is based purely on size
How does electrophoresis separate proteins?
By charges. Use a pH that will cause all of the proteins to be positive or negative except for the one you are trying to separate. Want the one you are separating to be a different charge than the others. Don’t want it to be neutral or it won’t separate out. Can be achieved by comparing the PI to the pH of the solution. If PI is lower than the pH charge will be negative. If PI is higher than pH it will be positively charged. If PI equals PH then it is neutral
What are the most prevalent extra cellular proteins?
Keratin, elastin, collagen.
What are the primary cytoskeleton proteins?
Tubulin and actin
What is myosin?
A motor protein
What type of receptors are hormones most likely to act on?
Enzyme linked receptors and G protein coupled receptors.
Not as likely to use ligand gated ion channels because hormones are found in small concentrations but have large effects due to second messaging.
Ligand gated ion channels do not use second messengers
Sodium and potassium are used for
Action potentials and are found in their free states rather than bound to proteins.
Chloride is
Readily excreted by the kidney
Calcium is normally found bound to a protein because
It must be sequestered in the bloodstream and intracellularly because calcium is used for muscle contraction, exocytosis and many other cellular processes that must be tightly regulated
What are characteristics of antibodies?
Label antigens for targeting by other immune cells
Antibodies can cause agglutination by interaction with antigen
Have to heavy chains and two light chains.
DO NOT BIND TO MORE THAN ONE DISTINCT ANTIGEN
What ion channels are responsible for maintaining the resting membrane potential?
Ungated channels.
What are the components of all trimeric G proteins?
Galpha, Gbeta and Ggamma
Gs, Gi and Gq are subtypes of the Galpha subunit of the trimeric G protein and differ depending on the G protein coupled receptors function
Ion-exchange chromatography
Separates ions and polar molecules based on their affinity to the ion exchanged. Works on almost any charger molecule including large proteins, small nucleotides and amino acids.
Doesn’t work when pI is very close
Size exclusion chromatography
Molecular sieve,
Molecules in solution are separated by their size and in some cases molecular weight. Usually applied to large molecules such as proteins
Isoelectric focusing
Electrophoresis technique that separates proteins based on their isoelectric point (pI). The pI is the pH at which a protein has no net charge and does not move in an electric field
Native PAGE
Also known as a CN page. Separates acidic water soluble and membrane proteins in a polyacrylamide gradient gel.
Doesn’t use a charger dye so the electrophoretic mobility of proteins is related to the intrinsic charge of the proteins
Difference between gel and chromatography
Gel can only handle a small volume of protein so chromatography is used to separate larger volumes
How does the gel for isoelectric focusing differ from the gel for traditional electrophoresis?
The gel in isoelectric focusing uses a pH gradient. When a protein it in a region with a pH above its pI it is negatively charged and moves toward the anode. When it is in a pH region below its pI it is positively charged and moves toward the cathode. When the pH equals the pI the migration of the protein is halted.
Anode is positively charged
Cathode is negatively charged
UV spectroscopy
Best used with conjugated systems of double bonds. Things such as aromatic amino acids will be adequate for UV absorption
Affinity chromatography
Protein elites off of an affinity column by binding free ligand.
If the binding is not reversed the free ligand competes with the active site of the enzyme which lowers its activity
Bradford protein assay
Spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composition of the measured proteins.
The Bradford reagent (is blue) forms a complex with proteins in solution which shifts its absorption maximum from 465 to 595nm. The absorption is proportional to the amount of protein present
What property of protein digesting enzymes allow for a sequence to be determined without fully degrading the protein
Selectivity.
The selective cleavage of proteins by digestive enzymes allow fragments of different lengths with known amino acid endpoints to be created. Allows us to make a basic outline of the amino acid sequence
What developmental stage had the greatest nuclear to cytoplasmic ratio?
Blastula
Morula stage
Early embryo stage consisting of 16 cells (called blastomeres) in a solid ball contained within the zona pellucida
Mulberry
The blastula is formed from this
Blastula
Hollow ball of cells, referred to as blastomeres, surrounding an inner fluid filled cavity called the blastocoel
Zygote
A diploid cell resulting from the fusion of two haploid gametes. A fertilized ovum
