BCMB 3100 Test 2

Vmax

Vmax

def: maximum velocity when all enzymes are saturated with substrate

dependent on enzyme concentration

kcat (K2)

turnover number which is achieved at Vmax

rate constant of rate-limiting step of the reaction

Kcat = Vmax/ [E]total (at high [S], [E] total would be all in ES complexes so really its Vmax/ ES concentration)

Km

stability of enzyme-substrate complex

compilation of rate constants: Km = (K-1+K2)/K1 (ES falls apart/ES forms)

independent of enzyme concentration

catalytic efficiency

=Kcat/Km

how fast the ES makes product

how easily ES is formed

larger = more efficient enzyme

Michaelis-Menten Plot (how to find Vmax and Km)

Vmax is the asymptotic value that the MM plot attempts to reach at high [S] values

Km is the [S] at Vmax/2

hallmark behavior of saturated catalyst in MM plot

starts as a first order with respect to [S] (linear phase) and then becomes a zero order at high [S] because rate stops depending on [S]

at high [S] values all enzymes are utilized in ES complexes

at high [S], V0 = K2 x [ES]

how to calculate Vmax and Km on Lineweaver-Burk plots

1/Vmax= y-int

slope= Km/Vmax

irreversible v reversible inhibitors

Irreversible- inhibitor covalently binds to groups on the active sites of an enzyme

reversible - inhibitor noncovalently binds to site on enzyme

3 types of reversible inhibitors

competitive: attaches at active site

• Vmax: unchanged

• Km: increased -can be overcome by increase [S]

noncompetitive: attaches at allosteric site -Vmax: reduced -Km: unchanged -cannot be overcome by increasing [S]

uncompetitive: attaches at group of the active site -Vmax: reduced -Km: reduced -cannot be overcome by increasing [S]

anabolism vs catabolism

anabolism- creating molecules from smaller components; biosynthesis - example: gluconeogenesis

catabolism- breakdown of larger molecules into simple components - example: glycolysis

Redox reactions

reactions in which one species loses an electron and another gains it

LEO says GER

electron carriers in metabolism

NADH and FADH2

help power metabolic pathways - redox reactions ; coupling reactions

Reduced and oxidized forms of NAD and FAD

NAD: reduced: NADH oxidized: NAD+

FAD: reduced: FADH2 oxidized: FAD

Comparing single carbon molecules based on free energy of oxidation, stability, energy, and usefulness as fuel

more energy= better as fuel, higher free energy of oxidation, less stable

why is ATP the energy currency in biology?

ATP hydrolysis is an energetically favored reaction with a negative delta G

ATP also has a very high phosphoryl transfer potential which means it is very likely to give up a P and release energy in the process -(due to electrostatic repulsion, stabilization due to hydration, increase in entropy, and resonance stabilization)

based on the delta G, which steps are irreversible

steps 1, 3, and 10

identify redox reactions in glycolysis

step 6 - glyceraldehyde-6-phosphate --> 1,3-bisphosphoglycerate and NAD+ +Pi --> NADH + H

identify a step that shows phosphoryl transfer potential

step 7- 1,3-bisphosphoglycerate --> 3-phosphoglycerateand ADP--> ATP

1,3-bpg has a higher phosphoryl transfer potential than ATP so it gave up a P to create ATP

identify a step that shows substrate level phosphorylation

step 7- 1,3-bisphosphoglycerate --> 3-phosphoglycerateand ADP--> ATP

step 10- phosphoenolpyruvate (PEP) --> pyruvate and ADP-->ATP

explain how coupling facilitates thermodynamically unfavorable reactions (ATP hydrolysis and oxidation driving les favorable reactions)

when one reaction is not thermodynamically favored it can be coupled with one that is greatly favored because delta Gs are additive and the excess negative delta G can cancel out the positive delta G

this means that the free energy released from the thermodynamically favored reaction can fuel the thermodynamically unfavorable reaction and still have excess that would make the whole coupled system favorable

ex. ATP is a high energy molecule with a high phosphoryl transfer potential as well as a decreased stability born out of the electrostatic repulsion of the three negatively charged phosphate groups next to each other. For this, ATP hydrolysis is an energetically favored reaction and is typically coupled with reactions like glucose phosphorylation to drive it

redox and thioester in glycolysis

oxidation is an energetically favored reaction as the removal of an electron leaves the molecule in a lower energy, more stable state

step 6 of glycolysis shows a redox reaction that uses a thioester as an intermediate to facilitate a coupled system. In the reaction, energy from the oxidation step is stored in the thioester intermediate which is a high-energy molecule which helps drive the unfavored step by providing a molecule which's reactivity is energetically favored and will drive the reaction to create the final product of 1,3-bisphosphoglycerate

explain substrate level phosphorylation and why 1,3-bisphospglycerate and phosphoenolpyruvate are substrates

substrate level phosphorylation involves attaching a phosphate group to an ADP molecule to create ATP

both 1,3-bisphosphoglycerate and phosphoenolpyruvate are substrates of these reactions because they have a higher phosphoryl transfer potential than ATP and will readily give up a P to attach to ADP

PET scan and how it detects cancer

PET scans use slightly radioactive sugars that accumulate in areas of high glycolytic activity (as is common with cancerous tissue due to excess proliferation) and release gamma rays that are then detected with a machine. Since there is a lot of sugar accumulation in cancerous tissue, there is a stronger concentration of gamma rays being released from that location and will result in a bright spot in the PET scan

Warburg effect (glucose metabolism in normal cell v cancerous cell)

while normal cells typically use mitochondrial oxidative phosphorylation to generate ATP, cancerous cells rely on glycolysis even in the presence of oxygen which is why cancerous cells take up more of the radioactive sugar

Given the pathways of glycolysis and gluconeogenesis, identify which reactions are most energetically favorable in one pathway under typical physiological conditions, and thus must be bypassed in the opposing pathway

steps 1, 3, and 10 of glycolysis are thermodynamically favored to the point that it is considered irreversible. These steps must be bypassed during gluconeogenesis

identify the three ways that metabolic pathways are regulated

1. compartmentalization: controlling where the substrate is in relation to enzyme- product of one reaction is the substrate of next and much be transported to another organelle where the next enzyme is

2. regulation of gene expression: many points in the process for protein synthesis. ex. DNA transcription regulation; not as much enzyme created

3. regulation of enzyme activity: achieved through allosteric enzymes (R vs T (biologically inactive) states) and reversible covalent modification

describe the role of allosteric enzymes in the regulation of metabolic pathways

regulate the flux of biochemicals

forces enzymes in the T or R state based on regulation

PFK2/FBPase2 bifunctional enzyme pattern

serine phosphorylated= PFK2 inactive / FBpase2 active (enzyme dephosphorylates substrate and activates gluconeogenesis)

serine dephosphorylated = PFK2 active / FBPase2 inactive (enzyme phosphorylates substrate and activates glycolysis)

define flux and how flux of metabolic pathways is affected by allosteric regulation

all of the enzymes in a particular pathway will be affected equally by allosteric regulation - this causes a flux (consequential increase or decrease in pathway activity throughout the pathway)

allosteric regulation can be inhibitory or excitatory. If inhibitory, enzyme will be in the T phase while excitatory will set the enzyme in an R phase

for example, increased PFK2 will have an increased flux effect on glycolysis

R/T states; allosteric effect on RT

enzymes in equilibrium between relaxed (active) and tense (inactive) state

allosteric inhibition fixes enzyme in T state and allosteric activation fixes enzyme in R state

how and why AMP and ATP allosterically regulate flux of glycolysis and gluconeogenesis

ATP is the allosteric indicator of PFK1/1,6-FBPase activity When ATP predominates, it attaches at the regulatory site and phosphorylates the serine which then causes an increased flux in gluconeogenesis by having 1,6-FBPase dephosphorylating fructose 1,6-bisphosphateWhen AMP concentration predominates, the PFK1 section of the enzyme is activated which causes an increased flux in glycolysis and decrease in gluconeogenesis. This causes the dephosphorylated enzyme to start phosphorylating fructose 6-phosphate to 1,6-bisphosphate

energy charge

[ATP] + 1/2 [ADP]

[ATP] + [ADP] + [AMP]

closer to 1 = ATP utilizing pathways closer to 0 = ATP generating pathways

importance of reciprocal regulation of glycolysis and gluconeogenesis and role of allosteric enzymes in regulation

Both pathways are reciprocally regulated as to maintain blood sugar levels and energy substrate concentration within the cells

ATP works as a negative feedback indicator whether PFK1 should be activated to create more ATP or F1,6Bpase in an excess ATP environment to create glucose

reciprocal enzyme activity of glycolysis and gluconeogenesis

when one pathway is highly active the other is inhibited; gluconeogenesis uses more ATP molecules than glycolysis produces so if both were to be activated at the same time, the net effect would be a depletion of ATP. Both glycolysis and gluconeogenesis are regulated by ATP concentration which acts an an allosteric regulator

Certain enzymes of the pathways are allosterically affected by both AMP and ATP or just one or the other. Regardless the flux they cause affects the entire pathway

what are coenzymes

small organic cofactor derived from vitamins that support enzyme activity

biotin and gluconeogenesis

biotin binds to CO2 within gluconeogenesis

identify the steps of gluconeogenesis that are regulated by compartmentalization

Step 1: the first substrate that is used in gluconeogenesis, oxaloacetate, cannot pass through the mitochondria membrane so it is reduced into malate which can pass through the membrane through a malate protein channel and is then oxidized in the cytoplasm

steps in middle: occur in the cytoplasm

step 11: glucose is synthesized in the endoplasmic reticulum

compare and contrast the effect of insulin and glucagon on glucose metabolism in the fasting and fed states

fasting state: blood sugar level low --> increased glucagon (catabolic) --> glycolysis, glycogenolysis increased

fed state: blood sugar level high --> increased insulin (anabolic) --> gluconeogenesis, glycogen synthesis increased

vice versa

explain how bifunctional enzyme PFK2/FBPase2 controls the level of the PFK1 allosteric regulator fructose 2,6 bisphosphate. integrate with insulin/glucagon signaling

PFK2 uses some fructose-6-phosphate to create F-2,6-BP which is a potent activator of the enzyme PFK1 which facilitates glycolysis

FBPase2 is an inhibitory enzyme that reduces concentration of F-2,6-BP and increases activation of F-1,6-BPase which converts fructose-1,6-bisphosphate to fructose-6-ohosphate and facilitates gluconeogenesis

in states of high blood sugar levels, Insulin will be released and PFK2 will be activated to catalyze glycolysis PFK2 will use some fructose-6-phosphate to create fructose-2,6-bisphosphate which will highly catalyze the activity of PFK1 to turn fructose-6-phosphate to fructose-1,6-bisphosphate for glycolysis

in states of low blood sugar levels, glucagon will be released and FBPase2 will predominate and decrease the concentration of F 2,6 bpase and F1,6BPase will activate to catalyze gluconeogenesis

when the PKF2 domain of the bifunctional enzyme is phosphorylated, gluconeogenesis is activated due to the increased activity of fructose-1,6-bisphosphatase when the PFK2 domain is not phosphorylated, glycolysis prevails as PFK2 is active

signal, receptor, signaling effectors, and short/long term effects in insulin signaling pathway

**application question

signal: insulin receptor: tyrosine kinase signaling effectors: kinase cascade (signal amplification) short term effect: cellular movement OR enzyme activation long term effect: altered DNA transcription

kinases v phosphatases

kinases - phosphorylate (Take P from ATP/GTP and attach it to substrate)

phosphatases- dephosphorylates (takes P from substrate and removes it w water molecule)

how does insulin signaling pathway affect GLUT4 location in the cell

a messenger protein released from the insulin receptor complex separates and activates cellular movement by taking vesicles with GLUT4 transports to the cell membrane and inputs the GLUT4 channels into the cells membrane

cytoplasm --> cell membrane

Glycolysis and gluconeogenesis are reciprocally regulated. The committed step in glycolysis, the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate is catalyzed by the enzyme (PFK1/fructose 1,6 bisphosphatase). This type of enzyme is a (kinase/phosphatase) and its function is to phosphorylate its substrate, using ATP as the phosphoryl donor. This enzyme's activity is greatly stimulated when (fructose 6 phosphate/fructose 1,6 bisphosphate/fructose 2,6 bisphosphate) binds to an allosteric site as a potent activator, resulting in increased flux through glycolysis. Two enzymes control the amount of this molecule in the cell. (PFK1/PFK2/FBPase2/ F1,6 BPase) is the enzyme that makes it, by phosphorylating fructose 6-phosphate. It does not phosphorylate all of fructose 6-phosphate: only a little because this is such a potent activator. When glycolysis needs to slow and gluconeogenesis predominates, then this enzyme is active: (PFK1/PFK2/FBPase2/F1,6BPase), dephosphorylating this potent activator back to fructose 6-phosphate.

PFK1 kinase fructose 2,6 bisphosphate PFK2 FBPase2

explain type 1 diabetes with insulin signaling pathway

type 1 diabetes is a autoimmune disease that is caused due to a decreased production of insulin from pancreatic cells

because of the lack of insulin, the signaling pathway within the cells is decreased meaning that GLUT4 channels are not placed into the cell membrane with required efficiency so sugar tends to stay in the blood stream

identify the subcellular locations where each step of glycolysis and gluconeogenesis occur in a liver cell. integrate both pathways

all of glycolysis occurs in the cytoplasm

step 1 of gluconeogenesis occurs in the mitochondrial matrix
step 2-10 of gluconeogenesis occurs in the cytoplasm (overlap with glycolysis- think bifunctional enzyme for fructose-6-phosphate