BIOCHEM UW: Enzymes

Question 1

Transcription Factors

Answer 1

• bind to DNA & alter transcription levels by affecting RNA polymerase binding

Question 2

• “PEPCK catalyzes the formation of PEP” during which part of metabolism? by decarboxylating oxaloacetate & transferring a phosphate group from GTP.
• What is the balanced equation and what is decarboxlyation?

Answer 2

• during gluconeogenesis
• decarboxylation: CO₂ is removed from OAA (reactant), yeilding CO₂ as a product of the reaction
• GTP (reactant) transfers a phosphate group to the decaroxylated OAA, producing GDP & PEP
• Remember, PEPCK IS AN ENZYME!! So it facilitates above ^^ steps but is not altered by the reaction-they never apear in the right or left of the reaction/product, but may be written above/below arrow to show participation
• Balanced equation will be: OAA + GTP–> GDP + CO₂ + PEP

Question 3

Null Hypothesis

Alternative Hypothesis

Answer 3

• The null hypothesis (H₀) theorizes that there is no difference between 2 groups,
• but the alternative hypothesis (H_A) rivals the null by supposing that a difference does exist.
• The significance of the results is then determined by whether a p-value is equal to or less than α, the predetermined level of significance
  
  • alpha is equal to 0.05, which means that p values ≤0.05 are considered statistically significant.

Question 4

Proteins can be degraded by __________enzymes

Peptide hydolysis:

Amide bond condensation:

Answer 4

• protease enzymes
• During peptide hydrolysis, a water molecule is used/consumed to cleave the C-N bond in the amide linkage using an acid (H+) or base (OH-)
• In contrast, the reverse of amide hydolysis is amide bond condensation, which involves the formation of water molecule from two amno acids to form a larger peptide

Question 5

is heat released or consumed when a substrate enters & binds to enzyme’s active site?

Answer 5

• heat is released from the formation of weak bonds (hydrogen, hydrophobic, ionic bonds) between 2 molecules

Question 6

Endergonic & Exergonic reactions

Answer 6

• Endergonic reactions are nonspontaneous reactions that absorb energy because their Gibbs free energy change ΔG is positive.
• Exergonic reactions are spontaneous reactions with negative ΔG values that release energy as they proceed.

Question 7

What is conformational stability? & What can it be measured by?

Answer 7

• the conformational stability of a protein is its ability to maintain its 3-D folded (tertiary structure) form & can be measured by the melting temp (T_m) at which half the proteins in solution are folded & half are denatured
  
  • ​_LOW T_M=DECREASED CONFORMATIONAL STABILITY_ becuase less thermal energy is required to denature the protein
• conformational stability does not necessarily affect protein function, as long as the protein is maintained at a temp that allows for proper folding
  *

Question 8

Turnover number K_cat

Answer 8

• ability of a folded enzyme to catalyze a reaction is measured by the turnover number, K_cat
  
  • which represents the number of reactions catalyzed per second per active site in solution

Question 9

Ternary enzyme complexes

ordered and random reactions

Answer 9

• one enzyme & two substrates
• ordered reaction: requires that substrates and products be bound and released in a specific sequence.
• For random reactions, the order in which substrates bind is not important and does not affect the progress of the reaction.

Question 10

Posttranslational modification

Enzymatic activity and inhibition

Answer 10

• enzymatic activity is dependent on the correct sequence & conformation of specific amino acids in the enzyme active site
• Mutating critical amino acids generally inhibits enzymatic function
• Deliberately mutating amino acids of interest can help reveal their role in enzymatic function

Question 11

pH & enzyme activity

Answer 11

• The activity of an enzyme is greatest at the optimal pH of that enzyme, and gradually decreases at pH values that are significantly lower or higher.
• Proenzymes or zymogens are the inactive forms of enzymes that require post-translational modifications to become activated.
• Proteolytic cleavage (proteolysis) is one such post-translational modification that involves the breakdown of proteins into shorter polypeptides or amino acids by enzymes known as proteases.

Question 12

Hill Coefficient / Cooperatively

Sigmodial / hyberbolic graph shapes

Answer 12

• The Hill coefficient n is a quantitative measure of cooperativity.
• Enzymes in which n > 1 display positive cooperativity (inc binding affinity) and have sigmoidal dependence on substrate concentration.
• Enzymes where n = 1 or n< 1 exhibit no cooperativity or negative cooperativity, respectively, and exhibit hyperbolic kinetics.

The Hill coefficient n is a quantitative measure of cooperativity obtained through kinetic analysis. It can be determined by fitting the data to a modified form of the Michaelis-Menten equation:

V₀=Vmax [S]ⁿ / Kmⁿ+[S]ⁿ

Question 13

Glucose uptake

Answer 13

• Insulin reduces blood glucose concentrations by increasing the rate of glucose uptake and glucose consumption.
• Glucokinase converts glucose into glucose-6-phosphate in the first step of glycolysis.
• Insulin release stimulates glucokinase and increases the production of glucose-6-phosphate.

Question 14

Michaelis-Menten Kinetics show which curve? hyperbolic or sigmoidal?

WHAT IS THE LIMITING FACTOR OF MICHAELIS-MENTEN?

Answer 14

• HYPERBOLIC CURVE!!
• ENZYME CONC IS THE LIMITING FACTOR, not the substrate!

Question 15

Glucose uptake in cells is:

Answer 15

• via passive transport (doesn’t require ATP)
• glucose enters cells down a conc gradient through the GLUT transporters

Question 16

Noncompetitive inhibition

Answer 16

• DEC V_MAX
• NO K_M CHANGE
• They bind the free enzyme and the enzyme-substrate complex with equal affinity.

Question 17

UNCOMPETITVE INHIBITOR

Answer 17

Question 18

Define V_max

Answer 18

• V_max is the maximum possible rate of an enzymatic reaction when a given concentration of enzyme is present
  
  • V_MAX is limited by the # of enzyme active sites
• in a michaelis-menton graph, [S] is significantly larger than [E] at every [S] measured. (however this doesn’t mean that all active sites are occupied–>Vmax is reached-it doesnt indicate that)
• However, Vmax occurs only when the concentration of substrate [S] is high enough that all enzyme active sites are bound
• “only when the enzyme operates at V_max is the substrate BOUND TO EVERY EZYME ACTIVE SITE IN SOLUTION”

Question 19

Thermophilic vs non-thermophilic enzymes

Answer 19

• Enzymes operate optimally at a temperature that is high enough for the enzyme to maintain flexibility but low enough that the enzyme does not denature.
• Most enzymes have evolved to operate optimally at the temperatures to which they are typically exposed in their host organisms.

the ability for the active site to adopt & maintain the correct shape is influenced by temperature:

1. At low temperatures, molecular motion slows down and the active site becomes inflexible. Under these conditions the active site cannot easily change shape to accommodate the transition state, so the reaction rate is slow.
2. At high temperatures, molecular motion speeds up and the intramolecular bonds that maintain the enzyme’s tertiary structure are broken. This causes proteins to denature, destroying the shape of the enzyme’s active site and halting catalytic activity.

to facilitate survival: enzymes have evolved to operate at the enviromental temperatures to which organisms are exposed

Question 20

Example: Thermophiles are microorganisms that thrive at temperatures above 40 °C. what charactersitcs can we conclude?

Answer 20

• According to the question, thermophiles are microorganisms that thrive at temperatures above 40 °C. Therefore, enzymes from thermophiles typically have optimal activity above 40 °C and most likely have a relatively low catalytic rate at room temperature (25 °C)
• Thermophilic enzymes must be resistant to thermal denaturation so that they can maintain their tertiary structures at the high temperatures of their natural environment

although enzymes from thermophiles typically operate slowly below 37 degress, this is nost likely due to inflexibilty of the active site, not denaturation

• Accordingly, thermophilic enzymes most likely maintain their tertiary structure below 37 °C because molecular motion at this temperature is too slow to break intramolecular bonds.

Question 21

Table of types of inhibitors

mixed inhibitors

Answer 21

• Mixed inhibitors have characteristics of both uncompetitive and competitive inhibitors, and may cause an increase, decrease, or no change (ie, noncompetitive inhibitors) in Km, depending on whether they favor binding to E or ES.
• all mixed inhibitors binds E & ES but may favor one over the other
• when a mixed inhibitor binds E and ES with equal affinity, it is a noncompetitive inhibitor

Question 22

Tie Gibbs free energy with metabolism (irreversible & reversible reactions)

Answer 22

• Whether a reaction will proceed is dependent on the Gibbs free energy change ΔG of that reaction.
  
  • The natural tendency of a reaction is to minimize the Gibbs free energy, so chemicals in a lower energy state (more stable) are more likely to form under a given set of conditions than those in high energy states.
• A negative free energy change (−ΔG) is associated with a favorable reaction that proceeds spontaneously (products have decreased free energy relative to reactants)
• Positive free energy change requires outside energy to drive the reaction to completion. (forms less products)
• Reactions with large free energy changes between the products & reactants, are essentially irreversible, and therefore unidirectional (because the reverse reaction is so unfavorable that it essentially never occurs)
  
  • example: synthesis of pyruvate from PEP

Question 23

Mixed inhibition table

mnomonic

Answer 23

• Inhibitors that have equal affinity for free enzyme and the ES complex represent a special case called noncompetitive inhibition.
• Noncompetitive inhibitors cause a decrease in Vmax but have no effect on Km. The name “noncompetitive” can be distinguished from the similar “uncompetitive” by remembering that “n” and “m” are adjacent in the alphabet, so “mixed” and “noncompetitive” go together.
• Binding free enzymes causes an increase in Km, binding ES complexes causes a decrease in Km, and when both are bound equally, Km is unaffected.

Question 24

Protein Ubiquination

Answer 24

• Proteins are tagged for degradation in a process known as ubiquitination, which causes the proteasome to recognize and degrade the marked protein via proteolysis (peptide bond cleavage).
• This system is responsible for degrading damaged or unnecessary proteins/enzymes.

Question 25

Loading controls on a gel

Answer 25

• Loading controls normalize protein detection and ensure that protein loading is standardized across the gel.
• Proteins used as loading controls tend to be ubiquitously expressed and have consistent concentrations across all cell/tissue types.
• Housekeeping genes are the most common loading controls
  
  • ​ example:α- and β-tubulin proteins are structural/mobile cytoskeleton components

Question 26

hydroxylation reaction

Answer 26

• Hydroxylation reactions replace hydrogen atoms with hydroxyl (–OH) groups and can occur in amino acids with aliphatic R-groups.
• Aliphatic amino acids are nonpolar, hydrophobic molecules with R-groups consisting of chains of C–C bonds that are single, branched, or in nonaromatic rings.

Question 27

Nucleophiles & electrophiles

Answer 27

Question 28

Enzyme purification

Answer 28

Question 29

phosphorylases

Answer 29

break bonds by adding Pi across them

Question 30

Enzyme specifity

Answer 30

• Different enzymes have different levels of specificity for their substrates.
• Enzymes with high specificity will modify only one molecule or a few highly similar molecules, whereas less specific enzymes modify several molecules within a particular class.
• An enzyme’s specificity can be determined qualitatively by observing whether it acts on similar substrates.

example: when a enzyme phosphorlyates pyrmidines U and T but not C EVEN THOUGH C IS NEARLY IDENTICAL, this indicates that the enzyme is specific to the aspect of U that differes from C. both T & U has 2 carbonyls, whereas C has only one!

Question 31

Catalysis

Cofactors & coenzymes

Answer 31

• Catalysis is the increase in a reaction rate that occurs when a catalyst decreases the activation energy of a reaction.
• Catalysts decrease activation energy by interacting with substrates to stabilize the transition state between reactant and product.
• Enzymes are biological catalysts (primarliy composed of amino acids but require other components), and some enzymes require cofactors such as metal ions for catalysis.

Question 32

Under saturating conditions, what increases/decreases?

Answer 32

• The rate of an enzymatic reaction is directly proportional to the concentration of enzyme present in the reaction.
• When the active sites on an enzyme are saturated, an increase in substrate concentration will not change the reaction rate (bc Vmax is already achieved/all active sites are occupied) but an increase in enzyme concentration will (by increasing the number of active sites present in solution), thus increases Vmax

Question 33

an increase in Km would increase what?

Answer 33

• Km is the concentration of substrate at which ½Vmax is achieved.
• An increase in Km would _increase the amount of substrate needed to achieve Vmax_ but most likely would not alter Vmax itself.

Question 34

reaction rate is calculated as: ?

a longer reaction time would result in: ?

Answer 34

• as the amount of product formed divided by the amount of time required for product to form
• A longer reaction time would result in more total product but would not increase the rate of the reaction.