lecture 13

Question 1

why are enzymes regulated?

Answer 1

cellular conditions change constantly altering the need for certain reactions so there is the need to turn on or off these enzymes accordingly or simply change the rate of product formation and also the unnecessary accumulation of or use of substrate

Question 2

what are the two basic mechanisms for the regulation of total enzymatic activity?

Answer 2

alter the amount of enzyme that is available and alter the activity of existing enzyme molecules

Question 3

how do we alter the amount of enzyme that is available?

Answer 3

remember from the Vmax=K3[E]T equation that if the amount of enzyme changes [E]T changes and so Vmax will decrease; this requires a rapid turnover of the enzyme which is not common and this is more a long term effect

Question 4

how would the altering of existing enzyme molecules occur?

Answer 4

through non covalent allosteric modulators that bind reversibly to a specific site but not the active site to affect activity; the other way is through reversible covalent modifications of the enzymes in which a small molecule covalently binds to the enzyme and alters its conformation and activity until it is removed by breaking the covalent bond

Question 5

compare and contrast feed-back inhibition and product inhibition

Answer 5

feed back inhibition is where the product of one enzyme in a pathway inhibits an enzyme earlier in the pathway and these often inhibit branch point enzyme; product inhibition is where the product of an enzyme inhibits an enzyme earlier in the pathway

Question 6

interpret the roles of these types of inhibition for multi-enzyme pathways

Answer 6

the metabolic pathways are a long sequence of enzyme catalyzed reactions in which the product of one reaction is the substrate for the next and so the flux of metabolites through the pathway is controlled by altering the activities of the enzymes that catalyze the reactions and so the key is to keep everything balanced without intermediate build up. Regulatory enzymes act at the rate limiting step for the production of the eventual product, usually the first committed step for production of the product and is usually far form equilibrium.

Question 7

what is key in the feedback inhibition loop?

Answer 7

balance

Question 8

what acts at the rate limiting step for the production of eventual product?

Answer 8

regulatory enzymes

Question 9

what are michaelis menton enzymes?

Answer 9

like myoglobin, single subunit enzymes where the enzyme binds substrate, the enzyme makes product and the product goes away. Allosteric interactions, no physical interactions, no subunits and generally have a hyperbolic path

Question 10

what are the allosteric enzymes?

Answer 10

A structural change at a site different than the active site will cause changes in the enzyme active site that will affect the catalytic activity of the enzyme.

Question 11

how does the T versus R model affect the sigmoidal velocity versus [S] curve for allosteric enzymes?

Answer 11

Relaxed = more flexible enzyme, less protein-protein contacts, less cooperativity, enzyme is more active. Tense = more rigid enzyme, more protein-protein contacts, more cooperativity, enzyme is less active. So with the activator binding in the T state on a site other than the substrate binding site, you greater affinity of the substrate to bind to enzyme and the velocity increases in low levels of substrate whereas with the inhibitor bound to substrate with inhibitor you have less affinity and so the slower the velocity; note that whether you are activated or deactivated that this a product of cell signaling, i.e.- insulin, glucagon are activated by signaling from liver regulating blood glucose levels via enzymes

Question 12

on the sigmoidal velocity versus [S] curve, does Vmax change? does anything else change

Answer 12

no; the S0.5 or the substrate level that gives you half maximal activity for the allosteric enzyme and so when you shift to left you have higher affinity

Question 13

what are the effects of allosteric modulators on affinity and maximum velocity?

Answer 13

affinity is changed but not the maximum velocity

Question 14

T/F, allosteric modulators can either increase or decrease the affinity of the enzyme for the substrate?

Answer 14

T, as result the reaction rate can be affected positively or negatively at a given substrate concentration

Question 15

what kind of binding occurs between the allosteric modulators and the enzyme?

Answer 15

the binding is non covalent and reversible and so this allows for rapid changes in enzyme activity

Question 16

how do positive and negative modulators influence the curve?

Answer 16

the positive curve, which shifts the curve to the left is the effect of the positive modulator (an activator) increases enzyme activity by decreasing cooperativity (less sigmoidal shape more hyperbolic shape) and decreases K0.5 (increasing affinity of the enzyme for substrate); the negative curve is the opposite in which you have a negative modulator decreasing enzyme activity by increasing cooperatively and increasing the K0.5

Question 17

allosteric modulator binding sites can be on the same subunit or different subunit from those that have the enzyme active site? name an example

Answer 17

T, cAMP dependent protein kinase (protein kinase A or PKA) which phosphorylates many proteins to activate or inactivate them

Question 18

How does the cAMP dependent protein kinase work as an allosteric modulator?

Answer 18

In the absence of cyclic AMP (cAMP), the regulatory subunit has very high affinity for the catalytic subunit. When C is bound to R, C has no activity at all. When cAMP binds to R, R changes its shape and that makes it lose its affinity for the catalytic subunit. C dissociates from R. Now C is completely active and does chemistry in the cell. Eventually, the cAMP level in the cell will fall, and cAMP will no longer bind to R. When cAMP comes off of R, R goes back to its original shape and binds C with very high affinity. This shuts C down so it is inactive again.

Question 19

the regulation by covalent modification cannot be reversible or irreversible, T/F?

Answer 19

F, it can be reversible or irreversible.

Question 20

what is the most type of regulation via covalent modification?

Answer 20

reversible covalent attachment of small molecules like phosphate, adenylate, methyl group. These are always reversible

Question 21

what is also another common type of regulation by covalent modification?

Answer 21

the removal of a part of a protein by a protease resulting in its activation, this is irreversible

Question 22

what is a specific type of irreversible covalent modification?

Answer 22

proinsulin, protein is in its inactive form and so highly specific and regulated cleavage is committed by proteases removing parts of the precursor that cause the protein to refold and this refolding activates the enzyme or in this the protein, unable to reverse back to its inactive state

Question 23

so you used proinsulin as an example of irreversible covalent modification, what is another example?

Answer 23

chymotrypsin, inactive form of chymotrypsinogen with additional folds to prevent exposure of the active site, and once released into the small intestine the change in pH causes a conformational change (clipping of amino acid residues) and now its on forever digesting dietary proteins.

Question 24

name an example of reversible covalent modification?

Answer 24

reversible phosphorylation like with glycogen phosphorylase and glycogen synthase that receive signaling form glucagon and insulin

Question 25

we talk about the concept of reversible phosphorylation as an example of reversible covalent modification, how does this relate to phosphorylation?

Answer 25

we the concept of phosphorylation as the addition of a phosphate group to an alcohol side chain and this can be done be a protein kinase like where we would see this with a transfer of a phosphate group from ATP to serine, threonine, tyrosine that could activate or inactivate that protein. The opposite holds true with a phosphatase enzyme which hydrolyzes the phosphate group from the protein.

Question 26

how does the addition of the phosphate effect the protein?

Answer 26

it alters the enzyme conformation slightly b/c of the bulky, negatively charge group

Question 27

glycogen phosphorylase is an example of what type of phosphorylation, reversible or irreversible? what does it do

Answer 27

reversible covalent modification and allosteric modulation, and when you phosphorylate it you go from being inactive to active so that you can break down glycogen to glucose when needed.

Question 28

glycogen synthase is an example of what type of phosphorylation, reversible or irreversible? what does it do

Answer 28

reversible, and when you phosphorylate it, it goes from active form to inactive form when blood glucose levels are low

Question 29

where can you find glycogen phosphorylase?

Answer 29

in the liver and skeletal muscles and they function differently, but enough alike to make comparisons

Question 30

how does glycogen phosphorylase work?

Answer 30

in the liver, when you have glucagon, you see glucagon binds cAMP, protein kinase A and phosphorylation happens and glycogen phosphorylase becomes active

Question 31

AMP binding is considered a positive allosteric modulator for glycogen phosphorylase, T/F?

Answer 31

true

Question 32

how does allosteric and phosphorylation control of glycogen phosphorylase in skeletal muscle work?

Answer 32

when running, you use ATP to drive muscle contraction and it becomes ADP and enzyme takes up phosphate from 1 ATP (ATP+AMP) and puts it on another ADP to produce ATP and AMP levels go up indicating body needs more ATP. Note glycogen phosphorylase has an AMP binding site and this signals the break down glycogen to glucose to facilitate glycolysis to produce ATP for the muscle

Question 33

what is the major signal in muscle and liver?

Answer 33

AMP is the major signal in muscle, phosphorylation is the major signal in the liver

Question 34

what is an example of a protein-protein interaction?

Answer 34

protein kinase A, calmodulin, G proteins

Question 35

since we mentioned G proteins, how do these functions? This is important for cell growth signaling pathway

Answer 35

G-proteins bind GTP, but when it has the GDP the enzyme accommodates this and goes into an inactive form and is kept there by an SOS protein for example. So in a nutshell, protein interaction between RAS-GDP (g-protein) is inactive state and kept there by SOS until signal is received, SOS lets go of RAS protein and RAS then swaps GDP with GTP and then this is recognized by another protein like RAF for example and binding occurs and active state, RAF hydrolyzes GTP to GDP and dissociation occurs between RAF and now Ras-GDP complex returns and binds with SOS; opposite in cancer, SOS becomes insensitive and control is lost

Question 36

what is important for SOS when cancer happens?

Answer 36

SOS becomes insensitive and so control is lost and G-Protein is active all the time

Question 37

what is an Isozyme?

Answer 37

isoforms of the same enzyme that catalyze the same reaction but have different amino acid sequences (effect: induces slightly different enzymatic properties) and so different regulation of the same enzyme occurs in different tissue type (One isoform in one tissue type may have an allosteric regulator that doesn’t affect the other isoform in a different tissue type)

Question 38

in terms of Isozyme, why is LDH important?

Answer 38

if we recall LDH, you can convert lactate into pyruvate and convert into ATP and depending on tissue and their needs you will have different combination of isoforms, which again is the same enzyme with slight difference in structure and so in the heart, it loves lactate to produce pyruvate and so LDH is present because of energy requirement, but in liver, different because it accommodates the functions of the rest of the body and takes what it needs

Question 39

why is the compartmentization of pathways important?

Answer 39

the metabolic pathways are contained within membrane bounded organelles

Question 40

allosteric enzymes can have their activities altered (increased or decreased), T/F?

Answer 40

true

Question 41

how can allosteric enzymes have their activates altered?

Answer 41

by small non-substrate molecules called allosteric modulators, also altered by protein-protein interactions

Question 42

what does allo mean? steric?

Answer 42

other; site

Question 43

allosteric enzymes can often be multi-subunit proteins, T/F?

Answer 43

T, one subunit can be a catalytic subunit and another is a regulatory subunit.

All subunits can be catalytically active, but are controlled by cooperative interactions

Question 44

in terms of plotting, what shape do allosteric enzymes make?

Answer 44

sigmoidal dependence of velocity on [S]

Question 45

in terms of plotting, what shape do Michaelis Menton enzymes make?

Answer 45

hyperbolic dependence, no allosteric modulators can affect these enzymes because they are single subunit enzymes whereas allosteric enzymes are multisubunit

Question 46

what is relaxed state again?

Answer 46

Relaxed = more flexible enzyme, less protein-protein contacts, less cooperativity, enzyme is more active.

Question 47

what is tense state?

Answer 47

Tense = more rigid enzyme, more protein-protein contacts, more cooperativity, enzyme is less active.

Question 48

Allosteric regulators affect the tertiary structure of the subunit that they bind to and therefore the quaternary structure of the enzyme, T/F?

Answer 48

T

Question 49

what is the function of the activator for allosteric enzyme? what is the effect of removing the subunit interaction on the enzyme?

Answer 49

Activators make it easier for substrate to bind => more activity!; it makes the enzyme look more like a michaelis menton enzyme, like PKA

Question 50

what is the function of the inhibitor for allosteric enzyme?

Answer 50

Inhibitors make it harder for substrate to bind => less activity!

Question 51

what does increasing contact do to cooperativity?

Answer 51

increases

Question 52

what does decreasing contact do to cooperativity?

Answer 52

decreases

Question 53

what is the significance of the allosteric modulators binding being non covalent and reversible?

Answer 53

it allows for rapid changes in enzyme activity

Question 54

what is the binding like for allosteric modulators? what do they change for substrate?

Answer 54

non covalent and reversible; affinity but not the maximum velocity