BIOC 384 - Exam 2
What is the energy required of 2 K+ ions across the membrane
+3.6 kJ/mol
What is the free energy required to transport of 3 Na+ ions across the membrane
+41.4 kJ/mol
Three types of enzyme-mediated reactions
- coenzyme-dependent reactions - metabolite transformation reactions -reversible covalent modification reactions -acid-base catalysis - specific acid-base - general acid-base catalysis
The enzyme active sites contributed to a decreased activation energy in ΔG
- sequestered micorenvironment - binding interactions between the substrate and the enzyme that facilitate the formation of the transition state - catalytic functional groups
Enzymes increase reaction rates
- stabalize the transition state - provide an alternate path for product formation - optimal substrate orientation + increaed local concentration
Key parameters of enzymes
-enzymes usually bind substrates with high affinity + specificity -substrate binding to the active site induces structural changes in the enzyme -enzyme activity is higly-regulated in cells
The ΔG for ATP hydrolysis in cells is about -50 kJ/mol. Therefore how many total ATP must be hydrolyzed by Na+-K+ transporter to transport 3 Na+ ions out of, and 2 K+ ions in to a cell
1 ATP, becasue the total energy needed for an ion exchange is +45 kJ/mol which needs ~1 ATP (-50 kJ/mol)
The value of (1) ___ is the (2) _____ concentration at which the reaction rateis (3)____ Vmax; a high value of Km means that an enzyme has (4)____ catalytic activity at a low substrateconcentration
1) Km, 2) substrate, 3) 50%, 4) low
(1) ______ is a positive effector of hemoglobinthat shifts the conformational equilibrium from the (2)_____ state tothe (3)_____ state, which increases oxygen binding; this is anexample of (4)________ control.
1) O2 2) T 3) R 4) allosteric
Transition State
1. ES 2. TS 3. EP 4. E+P 5. Substrate
Oxygen binding to the heme group in hemoglobin initiates a series of events that mediate cooperative binding of additional oxygen molecules to the hemoglobin tetramer. Put in order a. Fe2+ moves into the plane of the heme group b. F helix movement alters hemoglobin structure c. O2 binds to the Fe2+ atom in the heme group d. Proximal His is pulled closer to the heme group e. Fe2+ radius is reduced owing to shared electrons
3,5,1,4,2
What is the fold-purification of a target protein if after a three-steep purification scheme involving centrifugations, column chromatography, and isoelectric focusin you lost 20% of target protein compared to what you started with, and there is 2% of the total amount of protein reamining compared to what you started with
40 - fold
Based on the pH activity curve of an enzyme shown here, which statement below is the mostlikely explanation for the shape of the curve showing maximal activity at pH 5.5?Assume that there are only two titratable groups in the enzyme active site and notitratable groups on the substrate
A Glu residue must be deprotonated and a His resiude must be protonated
C < B < A
Based on the binding data shown in the graph, put the three proteins inorder from lowest to highest affinity for the ligand L
Which statement below correctly explains why histidine can function as both a proton donor andproton acceptor in the same reaction mechanism?
Because histidine has a pKa of 6.0, which means the imidazole group is readily ionizable at neutral pH
Why do enzyme inhibitors have structures based on proposed transition state conformations
Because inhibitors with the transition state conformation have the most interactions in the active site
Negative allosteric regulators
CO2, H+, 2,3-bisphosphogylcerate (BPG) function as a negative allosteric regulator that shift the R state toward the T state to decrease O2 binding to the heme group (homotropic effector)
Protein sequencing
Can be done chemically using a method called Edmond degradation in which each amino acid is identitiifed in a sequential manner. Overlapping oligopeptides can be sequences
Enolase
Catalyzed a dehydration reaction converting 2-PGA into PEP using both acid-base and metal ion catalysis, the Mg2+ ions in this reactions are necessary for ionic interactions with substrate
Define the different types of column chromatography
Column chromatography is a macromolecular separation technique that is essential to most protein purification strategies
Interpret the results of protease treatment followed by Edman degradation to determine the sequence of a peptide
Edman degradation is a protein sequencing method that uses chemical labeling + cleavage. By fragmenting a polypeptide into small fragments using differential protease digestion, it is possible to deduce the amino acid sequence of the entire protein
Which of the following statements best describes the requirement that must be met before K+ions can pass through the selectivity filter of the potassium channel protein?
Eight water molecules must be stripped from the hydrated K+ ion for it to pass through the channel
The enzyme pyruvate kinase is regulated by fructose-1,6-bisphosphate (F-1,6-BP), whichincreases k cat and lowers K m . What effect does F-1,6-BP have on the catalytic efficiency of pyruvatekinase and is F-1,6-BP a positive or negative regulator of pyruvate kinase?
F-1, 6-BP increases the catalytic efficiency and is a postive regulator of the enzyme
R state
Hemoglobin with bound O2 high affinity for O2
T state
Hemoglobin without O2 molecules low affinity for O2
A) 2º active symporter, B) 1º active transporter, C) 2º active antiporter
Identify each of the three types of transporters shown in the figure (1º = primary, 2º = secondary
Summarize the separation methods that can be combined into 2-D PAGE
Isoelectric focusing can be paired with SDS-PAGE to separate proteins on the bais of both pI and molecular mass using 2-D PAGE Isoelectric focusnig- separates proteins on the basis of charge as a function of pH, type of gel SDS-PAGE- Separates molecules based on charge and size that includes the detergent SDS
How is isoelectric focusing used to separate two proteins of equivalent molecular mass?
Isoelectric focusing is based on overall net charge in which a protein band is formed where pH = pI.
Passive + Active membrane transport
Mediated by membrane proteins
Outline the steps of solid-phase peptide synthesis
Method to generate oligopeptides of up to 25 amino acids using successive rounds of covalent linkage, washing, and deblocking to add amino acids one at a time to a resin-attached C-terminal amino acid. Once the oligopeptide is synthesized it is released from the resin.
T to R state
O2 binding to hemoglobin proteins involved protein conformation changes from the T state to the R state that alter the affinity of other subunits for O2 binding
F helix
O2 binding to the iron atom in the heme group results in movement of the F helix as the iron move into the plan of the heme, resulting in coformation change elsewhere
Positive effectors
O2 is a positive allosteric effector (T state to R state)
Positive allosteric effector
O2 is a positive allosteric effector of hemoglobin that shifts the conformational equilibrium from the T state toward the R state to increae O2 binding to the heme group (homotropic effector)
T state
O2 unbound forms the hemoglobin called the T state for deoxyhemoglobin low affinity
A polypeptide was digested by chymotrypni and trypsin and the resulting peptide fragments sequenced by Edman degradation. Use the following information to determine the sequence of the intact polypeptide. The c-terminal amino acids for the peptide fragments shown are Chymo: (Gly, Trp, Phe) and Tryp:(Lys Gly Arg) Chymotrypsin: MG, PCSKW, ECRLEF Trypsin: PCSK, LEFMG, WECR
PCSKWECRLEFMG
Which amino acid pair constitutes the hydrophobic patch on sickle cell hemoglobin that resultsin formation of protein oligomers with the Glu-6-Val sickle cell mutation? Does normal hemoglobin containthis same amino acid pair constituting a hydrophobic patch?
Phe 85/ Leu 88 ; yes
Chymotrypsin cleaves on the carboxyl side of ____ or Trp amino acid residues. The amino acid ____ has a central role in this _____ catalysis reaction mechanism. A kep step in the first part of the reaction sequence is the release of the ____ terminal fragment as a result of the ____ residue protonating the substrate
Phe, Ser, acid-base, carboxyl, His
Active Membrane transport
Proteins are of two types primary and secodary
Explain the migration process of gel electrophoresis and how marker proteins are used to estimate the apparent molecular mass of an unknown protein
Separation of proteins based on charge + size Approximate the molecular mass of a protein + to find out if the purified protein includes more than one polypeptide chain
The [T]/[R] ratio decreased and the oxygen affinity is increased
The Hb Yakima variation is caused by the mutation Asp-99-His, whichresults in a shift in oxygen binding as shown in the graph. What effect does theAsp-99-His variation have on the [T] / [R] ratio of Hb Yakima protein conformationcompared to Hb Normal and how does Asp-99-His alter oxygen affinity?
Would you expect the alpha and beta subunits of hemoglobin to have more, fewer, or the samenumber of hydrophobic amino acids on the surface than are present on the surface of myoglobin? Explain
The alpha and beta subunits would have more than myoglobin because of subunit-subunit interactions.
An impure enzyme cell extract is run through a size-exclusion chromatographycolumn. The activity of the 120 kDa enzyme is measured before and after the chromatography step. Theenzyme has more activity after the chromatography step. Which of the following is most likely true?
There is a 20 kDa inhibitor in the protein extract that is separated by the column chromatography step
A patient, rescued from a burning home, comes into the ER with suspected carbon monoxidepoisoning. Which of the following would you consider to be the best treatment strategy for the patient?
Treat the patient with 100% O2 gas
Compare and contrast X-ray crystallography and NMR spectroscopy as tools for determining protein structure
X-ray crystallography uses a focused X-ray beam directed at a protein crystal to obtain a diffraction pattern NMR spectroscopy used to determine the relative locations of atoms in a purified protein solution.
Isoelectric focusing
a 2-dimensional SDS-PAGE are other methods used to separate small amounts of
column chromatography
a common method used to separate proteins, gel filtration, ion exchange, and affinity chromatography
Transition state theory
a reactant must reach an energy level required for chemcial transformations before product can be formed, enzymes lower the activation energy
Chymotrypsin
a serine protease that cleaves proteins using a combination of acid-base + covalent catalysis, that amino acids serine, histidine, and aspartate from a catalytic triad that functions to convert the serine into a highly reactive nucleophile
Identify the correct label for A,B,C,D in figure
a) NADH b) NAD+ c) oxidized reactant d) reduced product
For an uncatalyzed reaction in which A<->B there is a forward rate constant Kf of 10^3/s, and a reverse rate constant Kr of 10^-5/s. In addition, the reverse rate constant in the presence of a catalyst, Kr(cat), is 10^3/s. Remember that Keq= Kf/Kr= K1/K-1. Answer the three-part question a) do catalysts change the Keq of a reaction b) what is the Keq of the uncatalyzed reaction c) what is the value for the catalyzed forward rate constant Kf(cat)? d) Is the formation of B favored or unfavored
a) No b) 10^2 c) 10^5 d) favored
You need to separate two proteins from each other; one is Protein X = 80 kDa, which has a netnegative charge at pH 7, and the other is Protein Y = 10 kDa, which has a net positive charge at pH 7
a) X b) X c) X d) Y
How do two a-helices at the constriction point of the Aquaporin 5 transporter protein preventH3 O+ molecules from forming a H+ wire that prevents the transport of H2 O molecules across the membrane?
a-helices have inverted dipoles that reorient H2 O molecules in the channel to break H 2 O/H2 O H-bonds.
Choose the list of proteins that represent examples from each of the major proteins classes a. structural protein b. genomic caretaker protein c. metabolic enzyme d. transport protein e. cell signaling protein
a. actin b. RecBCD c. enolase d. aquaporin e. protein kinase A
There are three main types of metabolite transformation reactions a) which type combines two substrated from a larger product b) which types involved the addition of H2O
a. condensation b. hydrolysis
In the ____ model of muscle contraction, the ____ filaments are made of myosin protein and the thin filaments are made of _____ and other proteins. These two filaments slide past each other during muscle contraction, ____ the distance between Z disk proteins. This ____ is initiated by the bind of ____ to troponin
a. sliding filament b. thick c. actin d. shortening e. contraction f. calcium
Active transport
aganist the gradient
structural proteins
are often assembled into polymers to form filaments that provide cell structure and facilitate movement
Allosteric effector molecules
bind to hemoglobin and induce conformation changes that shift the equilibrium between R state and T state to affect O2 binding to the heme group
Enzymes are
biological catalysts that alter reactions rates without changing the overall change in free energy ΔG or the equilibrium constrant Keq of the reactions
Protein identification
can be done using mass spectrometry in which peptide sequences are identified on the basis of a uniqu molecular mass as compared to a database of prodicted molecular masses done computationally
HMG-CoA Reductase
catalyzes the four-electron reduction of HMG-CoA to mevalonate a step in cholesterol biosynthesis, the reaction uses two NADPH molecules as coenzymes
Metabolic enzymes
chemical catalysts that lower the activation energy of a biochemical reaction to increasing the rate of product formation without altering the equilibrium constant Enolase
Metabolic enzymes
chemical catalysts that lower the activation energy of a reaction without changing the equilibrium constant or standard change in free energy
Protein purification
combination of ART and SCIENCE because proteins have unique physical and chemical properties that require trial and error methods
Thin filaments
consist of polymerized actin proteins that serve as binding sites for the myosin head during muscle contractions, thin filaments also contain troponin and tropomyosin
Thick filaments
contains hundreds of myosin protein molecules arrange tail to tail within the fiber with the myosin head domain sticking out, the protein titin anchors thick filaments to the Z disk
Olgiopeptide synthesis
done using a chemical method in which block and de-blocking reactive groups is automated
Ion-exchange
exploits charge difference between proteins
Affinity chromatography
exploits specific binding properties of the target protein to separate it from other cellular proteins that lack this binding function
Transport protein
function as selective pores to permit or transport polar metabolites across the nonpolar lipid bilayer
Myoglobin
has a single polypeptide chain and functions as an 02 storage protein in muscle tissue
Membrane-bound metabolic enzymes
imbedded in the inner mitochondrial membrane and chloroplast thylakoid membrane
Passive Transport
in the same direction as the gradient
Membrane receptor proteins
involved in transducing extracellular signals across the plasma membrane
acid-base
involves water
Myoglobin
is a monomeric protein that serves as an O2 storage protein in tissues
Hemoglobin
is an O2 transport protein that contains two a-globin subunits and two B globin subunits without O2 molecules is the T state, low affinity for O2 with bound O2 is the R state, high affinity for O2
Muscle contraction
is initiated by Ca2+ release in response to neuronal signaling which leads to conformational changed in troponin so that myosin can bind to actin, ATP is required for the complete actin-myosin reaction cycle
Genomic caretaker proteins
maintain the chemical integrity of genetic informtion stored in DNA and RNA
Structural proteins
often assembled into long filaments that form cytoskeletal structures involved in cell migration, chromosomal segregation, and muscle cell contraction Actin
Bacterial porin
proteins are passive transport proteins with high selectivity in the outer membrane transport acoss the inner membrane required traditional transporters (ABC)
Membrane transports
proteins inserted into membrane must be able to associate with the hydrophobic environment of the membrane, they do this by having nonpolar amino acids on the outside of the protein, facing the hydrophobic portion of the bilayer
Genomic caretaker
proteins maintain the integrity of genetic information encoded in DNA and control gene expression. DNA metabolizing enzymes are required for DNA replication, repair, and recombination. RNA metabolizing enzymes are required for RNA transcription, processing, and stability DNA polymerase
Cofactors and coenzymes
provide additional reactive groups to the enzymes active state that complememnt the limited chemitry of amino acid side chains, cofacotrs are inorganic ions whereas coenzymes are small organic compounds often derived vitamins
R state
relaxes state of an enzyme subunit high affinity state
Cell signaling proteins
respond to changes in the extracellular environment by undergoing conformational or chemical changes that regulate cellular processes. A common mechanism to control signal transduction in cells is the phosphorylation + dephosphorylation of signaling proteins Protein kinase A
Cell siganling proteins
responds to changes in the environment by and relay information abou these changes to other proteins
Transport proteins
span membranes + function as selective pores. Passive transporters allow molecules to diffuse down a concentration gradient, active transporters act as energy-dependent pumps that transport molecules against a gradient Aquaporin
Hemoglobin
tetrameric protein that transports O2 from lungs to tissues
Primary transporters
that use energy from ATP hydrolysis
Secondary transporters
that use energy from gradients
Enzyme kinetics
the quantative study of rates of chemical reactions catalyzed by enzymes using purified kinetics provides the knowledge to determine reaction mechanisms, effects of regulatory molecules and the effects of mutations
Michaelis-Menten enzyme kinetics
used to study first order reactions (S ->) under stead-stale conditions as a means to relate the initial velocity as a function of substrate
Gel filtration-
uses porous hydrocarbon beads to separate proteins on the basis of size
general acid-base catalysis
which the proton transfer involves a functional group