Biochem Exam 1

At Vmax, what will adding more substrate do to the rate of the reaction? What about adding more enzyme?

Adding more substrate will not increase reaction rate, whereas the addition of more enzyme has the potential to.

When in the cross-bridge cycle does the sarcomere shorten?

The sarcomere shortens due to the interactions between the thick and thin filaments. Myosin interacts with actin, changes it shape, and ultimately pulls the thin filaments towards the middle of the sarcomere (power stroke) and causes sarcomere shortening in muscle contraction.

What are 3 possible problems that could occur within a higher animal cell if thin filaments were not produced when the cell completed mitosis?

-Essential materials needed for cell growth and repair may not be able to be transported across the cell, eventually leading to cell death. -The cell may collapse in on itself and not be able to hold its structure, meaning the cell could not carry out its intended function. -The cell may not be able to contribute to a muscle contraction within a sarcomere, since thin filaments are essential within muscle contraction.

Which amino acids are helix breakers, and why? What part of the protein structure do helix breakers affect?

Alpha-helices are part of the secondary structure of proteins. Proline, asparagine, and serine are helix breakers. Their R groups prevent them from forming alpha-helices. Unlike the other amino acids, proline's N bonds with two carbons, preventing it from forming the alpha-helix hydrogen bond. For asparagine and serine, the N-H and O-H in the R groups are more likely to hydrogen bond than the typical N-H, disrupting the formation of an alpha helix.

Explain in detail how pH affects a chain of amino acids, taking into account specific structures and protein function.

At lower, more acidic pHs, the amino, carboxyl, and certain R groups can be protonated. At higher, more basic pHs, those groups may be deprotonated. Proteins have optimal pH ranges in which they function best. When the pH becomes too low or high for the amino acid chain, the protonation and deprotonation that ensues may cause it to denature, break its current bonds, and possibly form new bonds. Alpha helices and beta sheets may change, and the amino acid chain may not be able to fold properly and interact with other amino acid chains the way it should, resulting in the protein not to being able to carry out its functions.

If the protein insulin is 5.734 kDa, using the average molecular weight of an amino acid roughly how many amino acids are in insulin.

Average molecular weight of amino acid = 113 Da, 5,734 Da/113 Da = 50.74 or about 51 amino acids.

Why do animals go into rigor mortis (a state of very rigid muscles and joints) after death?

Because their muscle fibers remain permanently contracted due to the loss of ATP when an organism dies. ATP usually binds to allow for actin and myosin to dissociate from each other, but without ATP the animal's muscle fibers will remain locked in a contracted state. This is what creates the rigid state after death.

What role does entropy have in the process of lipids spontaneously forming bilayers?

Entropy plays a role in this process by allowing water to point away from fatty acids to become more ordered (which lowers the entropy)

Describe the affinity of myosin during the cross bridge cycle and its steps:

First, the myosin heads are bound to ADP +Pi, and have a high affinity for actin. Next, the myosin heads bind to actin resulting in a low affinity for ADP +Pi which causes them to be realeased inducing the power stoke. Before doing the power stroke the myosin heads have a low affinity for ATP. However, after the power stroke they have a high affinity for ATP. Once a new ATP molecule binds to the myosin heads they'll have a low affinity for actin causing myosin to detach itself from actin. Finally, ATP is hydrolized starting the cycle all over again.

Why is it that glucose (C6H12O6) cannot readily diffuse across the plasma membrane without a glucose transporter, while molecular oxygen (O2) can?

Glucose is a very large molecule (MW = approximately 180.2 u) and is very polar, which would prevent it from passing through the hydrophobic inner region of the plasma membrane by simple diffusion (the inner region is hydrophobic because it consists of long saturated and unsaturated fatty acid tails, which experience a strong, entropy-driven hydrophobic effect when in aqueous solution). -Oxygen is both small in size (MW = 16.0 u) and non-polar, so it is able to diffuse passively across the plasma membrane's hydrophobic inner region.

What will happen in the process of muscle contraction if the concentration of Calcium ions (Ca2+) are low?

If the concentration of Calcium Ions (Ca2+) is low, then the myosin will be unable to form a cross bridge with actin and therefore cannot successfully promote a power stroke later in the process. This is because, myosin will remain bound to ADP and Pi as the cross bridge will not form unless troponin can effectively bind to Ca2+. In normal muscle contraction with sufficient amounts of Calcium, the binding of Ca2+ to troponin causes a shape change which allows myosin to interact and form a cross bridge with actin.

What would the effect(s) on muscle contraction be if each troponin complex regulated one crossbridge as opposed to seven?

If there were more troponin complexes regulating an equivalent stretch of actin, a higher concentration of calcium ions would be needed to bind troponin C and allow myosin to bind to actin and form the crossbridge. It may also result in the contraction being more "jerky" and less smooth, as more actin molecules are being regulated independently of each other, so myosin could potentially bind some places but not others.

Relating to composition of fatty acids, why would it be beneficial to have higher amounts of unsaturated vs saturated fats in a hypothetical organism depending on the temperature of the environment in which the organism is found?

Membranes are fluid by nature, and the composition of fatty acids is a factor that contributes to membrane fluidity. An organism living in a frigid environment may have a membrane with many unsaturated fats due to the need for a more fluid membrane. The organism cannot survive if there is no rigidity in the membrane. furthermore, an organism living in a very hot environment may have a membrane with little to no unsaturated fatty acids. this organism's membrane will already be fluid due to the heat, and it may even need the presence of cholesterols to further increase membrane stability and decrease fluidity.

What happens to muscle contraction when there is an excess of calcium present?

Muscle contraction depends on calcium. Calcium, which is released from an action potential controlled by the nervous system must be present in order for the power stroke to occur. Calcium binds to Troponin-C which causes a shape change in which TnT pulls TnI off of actin, allowing for the binding of the myosin head to occur. if there was excess calcium, there would never be a lag in the removal of troponin from actin, thus the myosin heads would always be able to make a power stroke after completing the prior cross bridge cycle. There would be multiple muscle contractions one after another. Since calcium will not be the limit in preventing the cross bridge cycle, it now may depend on ATP levels to keep up with the constant muscle contractions, as well as ones fatigue.

What is muscle contraction regulated by?

Muscle contraction is regulated by calcium because calcium binds to troponin-C which causes a shape change allowing for myosin to associated with actin, which allows the myosin to create a power stroke, overlapping the filaments and shortening the sarcomere.

What role does myosin play in muscle contraction?

Myosin is a motor protein that packs together to form the thick filaments of muscle. Myosin heads sticking out from the thick filaments pull along the thin filaments to carry out muscle contraction. During the Crossbridge cycle, ATP hydrolysis fuels the binding and dislocating of myosin from actin by altering the myosin's affinity to actin. The "walking" movement that results from the binding and unbinding of myosin to actin is ultimately what causes muscle contraction.

There are many myofibril associated proteins that are found in striated muscle tissue. Two of specific examples of these proteins are Nebulin and Titin. What are the functional similarities and differences between these two proteins?

Nebulin and Titin both act as stabilizing structures of myofilaments by acting as molecular rulers for organization. The difference is that Nebulin determines the length of thin filaments, and Titin acts as a ruler for the organization of the myofibrils.

You have recently discovered a new enzyme but still don't fully understand the properties responsible for moderating its functions. You have collected enough data on a certain factor you believe to be a reversible inhibitor to chart a Lineweaver-Burk Plot. Before you plot your data, what are the three different mechanisms you are trying to differentiate between, and how will this plot help?

Setting up a Lineweaver-Burk plot will reveal the nature of the inhibitor through subtle changes between normal and inhibited enzyme function. These plots show the linear progression of enzyme function by depicting the reciprocal of the Michaelis-Menten equation, and from them we can deduce the Km and Vmax values. Km will be reflected in the x-intercept of the plot, where x = -1/Km Vmax will be reflected in the y-intercept, where y = 1/Vmax If the inhibitor is competitive we will see a change in Km but not in Vmax because it is competing with the substrate and decreasing the formation of the enzyme-substrate complex. If noncompetitive, we will see a change Vmax but not in Km because the inhibitor is not competing with substrate and thus not affecting the formation of the enzyme-substrate complex. If we see a change in both we can assume the inhibitor functions through mixed noncompetitive inhibition.

Why are strong acids or bases poor buffers?

Strong acids and bases completely de ionize, buffers would have percentages of de ionization, it would not completely disassociate. Buffers need to ionize or disassociate very little

Transmembrane proteins can also have alpha helix structures, how could the "kinks" created by helix breakers be functionally advantageous?

The "kinks" from helix breakers like proline creates weak points in the helix. This structural diversity in the membrane could be useful in facilitating movement for transmembrane transport channels.

Thinking about the hydrophilic/hydrophobic character of the lipid bilayer, what makes beta barrels (essentially "rolled" beta sheets) a viable structure for transmembrane proteins?

The "rolled" structure of beta barrels allow the non polar amino acids to face outwards, thus interacting with the hydrophobic lipid tails of the membrane. Then, the polar amino acids can face inwards, creating a hydrophilic interior where hydrogen bonding can take place.

Why are proline, asparagine and serine considered helix breakers?

The R groups disrupt the hydrogen bonding that usually stabilizes helices. Pro- R group binds to NH2 group so it does not have normal geometry Asn/Ser- Hydrogen bonding on R groups is preferred, R groups length and nature make protons bind to them instead

Buffers are important in biological systems as they resist changes in pH. The buffering capacity refers to this ability of a buffer to resist changes in pH. What would happen to the buffering capacity if the concentration of the acid and its conjugate base were both doubled?

The buffering capacity would also double meaning that it would have a greater capacity to resist pH changes. The increased buffering capacity indicates that if more acid or base were added to the system there would be a smaller change in pH than at the original buffering capacity.

Explain the cross bridge cycle, what is responsible for regulation of the cross bridge cycle?

The cross-bridge cycle happens in our muscle cells and Myosin, which is also bound to ADP and a phosphate, starts in a cocked position then binds to actin and form a bridge. When this bridge forms a power stroke happens and as that power stroke is happening Myosin releases the ADP and phosphate. Next, a molecule of ATP attaches to the myosin and causes myosin to detach from actin. The new ATP that is attached to myosin then becomes hydrolyzed and becomes an ADP and phosphate like in the first step. This causes the Myosin to return to its original cocked position. When it is in this position the cycle is able to start again. This process is initiated and regulated by free calcium ions binding to the protein complex troponin. Calcium ion concentrations are increased when action potentials travel through the nervous system to motor neurons. These motor neurons then signal the sarcoplasmic reticulum to increase free calcium ion concentration. When calcium ions are bound to troponin, the active site of actin is then exposed.

What is the hydrophobic effect?

The hydrophobic effect occurs when water interacts with nonpolar substances. Water is polar, and cannot interact with nonpolar substances. When this happens, water molecules can only hydrogen bond with other water molecules in the hydrophobic system. This will lead to constraint, which is against entropy. The hydrophobic effect is a major driving force in the folding of proteins. Furthermore, we see the hydrophobic effect when lipids spontaneously form bilayers, or other similar structures, in an aqueous environment because their polar head groups will H-bond with water, but the nonpolar tails drive the water away. This forces the water to become more ordered around the tails, which causes constraint- the water molecules are constrained to face away from the tails and hydrogen bond with other water molecules. This is not favored, and decreases both entropy and stability in the system.

In a phospholipid bilayer there is a transmembrane protein that contains transmembrane helices. What effect would isoleucine being substituted with serine have upon the protein and the membrane?

The internal environment of a phospholipid bilayer is nonpolar so the amino acids in a transmembrane protein that are exposed to the nonpolar environment must also be nonpolar. Isoleucine is a helix-forming, nonpolar amino acid that sustains the H-bonding that upholds the helix backbone. The helix that is created will shield the polar amino acids from the nonpolar environment. If isoleucine was substituted with serine, the helix would break because there is more likely an H-bond to form with the R group than with the oxygen in the non variable region of amino acids. The protein then would lose its helix conformation and expose polar amino acids like serine to the nonpolar environment. This substitution would then disrupt the fluidity of the membrane because the polar amino acids and nonpolar phospholipids cannot interact.

What amino acids in this sequence may result in helix breakage? Which could promote beta turns? Draw a reasonable illustration of what the secondary structure of these amino acids might look like. Justify your reasoning for the structure you draw. Why can't we predict with certainty what the secondary structure will look like? ATYCHLAVMTWFIPNVTYHCAMWFQYDEHKPGPTYHCVWQDEECDFTGPGWQTCVWYYILLKYY

The italicized amino acids are likely to be helix breakers. The bolded amino acids are likely to promote beta turns. There are many possible ways to draw this secondary structure, and a rational explanation of the role the helix breakers and beta-turn promoting amino acids in the structure drawn by the student will be sufficient to justify their structure. It is extremely hard to predict the secondary structure of a region of a protein without experimental data because the actual secondary structure of proteins is highly dependent on the most energetically favorable tertiary structure of the protein. This means that a less energetically favorable conformation in one part of the protein might be required in order to reach the most stable conformation for the protein as a whole.

What is the reason that certain "preferred" angle pairs are most likely to occur in proteins, as found by Ramachandran?

The peptide bond between amino acids creates a planar conformation, which limits the possible ways in which a protein can fold. Certain angle pairs cause steric crowding, while the "preferred" angle pairs do not cause as much steric crowding and are far more likely to occur.

What are the driving factors for the polymerization of thin, thick, and intermediate filaments?

The polymerization of both the thin and thick filaments is an energy dependent process as the actin thin filaments require the hydrolysis of ATP to induce a change in the G-Actin to allow it and make it more energetically favorable to polymerize into F-Actin. The same idea is also true for microtubule thick filament polymerization as the process is dependent on the hydrolysis of GTP. On the other hand, the polymerization of intermediate filaments is driven by entropy. Specifically, the presence of a hydrophobic strip of AA in a single amino acid chain drives the formation of a coiled coil and thus the association of two chains to create a quaternary protein where the overall energy is significantly lower than if the hydrophobic strips were exposed. Because of this hydrophobic driving effect and not the hydrolysis of a compound like ATP or GTP, the polymerization of intermediate filaments is not classified as an energy dependent process.

Describe, in detail, the role of tropomyosin in muscle contraction.

Tropomyosin is essential in the controlled regulation of the process of muscle contraction because it is an extension of the troponin complex. This complex blocks the myosin binding sites on actin filaments to prevent involuntary binding and contraction. Since these complexes only occur every 7 actins, tropomyosin's role is to extend across and block the myosin binding sites on the 6 actin filaments in between these complex locations, further preventing uncontrolled muscle movements. Once substantial Ca2+ is released and bound to the troponin C, the tropomyosin moves away from these binding sites in unison with the troponin complex, allowing for myosin-actin binding on the 6 intermediate sites and therefore, muscle contraction.

What are the distinguishing properties that make the amino acids proline, asparagine, and serine alpha helix breakers?

Ultimately, the helix breakers disrupt the hydrogen bonding that is involved in the typical alpha helix formation, providing stability in the secondary structure. Whether it is due to proline's folding of the R group back onto the backbone, or the hydrogen bonding of the other two helix breakers. The helix breakers disrupt the hydrogen bonding and the hydrogen bonding is more likely to occur at the R groups of the helix breakers than it is at the carbonyl of the backbone.

What types of acids and bases make an effective pH buffer?

Weak acids and weak bases make effective pH buffers. This is because buffers need to ionize or dissociate very little so they can resist changes in the pH of the solution. Strong acids and strong bases make poor buffers because they completely deionize.

What causes micelles, vesicles, and bilayers to form?

When the concentration of lipids is greater than the critical micelle concentration, driven by the hydrophobic effect.

How is muscle contraction connected to the nervous system? Detail the pathway from your thoughts to your muscle ultimately contracting.

When you think about flexing a muscle, your nervous system generates a signal called an action potential. The action potential travels along neurons to the muscle of interest, resulting in a depolarization and a release of calcium ions from the sarcoplasmic reticulum. The calcium ions bind to troponin C, causing a shape change that moves troponin I off of the myosin binding site on every seventh actin and the tropomyosin off of the binding sites in between, allowing for the myosin heads of the thick filaments to bind to the actin of thin filaments. The cross-bridge formation that results leads to the release of ADP and Pi and a subsequent power stroke that induces the sliding mechanism of actin and myosin filaments. Ultimately, we see sarcomere shortening and thus muscle contraction.

What are the two primary forces which drive the folding of polypeptide chains?

constraints of the peptide backbone, (2) the "protection" of non polar amino acids (the hydrophobic effect= entropy)

If a particular inhibitor's ability to bind to an enzyme is reversible, what does this tell us about the type of bonds that are created between the inhibitor and enzyme?

the bonds are non-covalent, allowing for reversibility.