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