BIOC 221 - Lab Exam Flashcards
In the catalytic process, metal ions act as?
electrophiles
Metal ions help enzymes in what ways? (3)
1) aid in binding of S
2) accept/donate e’s
3) withdraw e’s to change partial charge distribution in S
The ability of certain metals to bind multiple ligands in their coordination sphere enables them to participate in? (2)
1) binding S and coenzymes to enzymes
2) polarizing reactive groups in active site
Example of a metal ion that contributes to the polarization of a functional group?
Zinc in Alcohol Dehydrogenase contributes to polarization of alcohol group
Role of Mg ions in many enzymatic reactions?
essential role in the binding of negatively charge phosphate group of ATP
Phosphatases - broadly classified into (3) groups depending on pH optima of the enzyme:
1) acid
2) neutral
3) alkaline Pases
What is Alkaline phosphatase? (what type of enzyme? uses?)
- non-specific hydrolase
- uses H2O as second substrate for a number of phosphate monoesters including PNPP (p-nitrophenyl-P) to produce yellow p-nitrophenyl & Pi
What is Alkaline phosphatase? (what type of protein?)
- glycoprotein with dimer of 2 similar sub units, each containing different binding sites for Zn(2+) and Mg(2+) that are required for catalysis
cofactors
inorganic ions (Fe2+, Mg2+, Mn2+, Zn2+)
coenzymes
complex organic/metalloorganic molecules
ex. NAD+
Some enzymes require both of what (2) things for activity? give an example.
require both a coenzyme & 1+ metal ions
alcohol dehydrogenase
prosthetic group
coenzyme or metal ion that is bound to enzyme
holoenzyme
enzyme with bound coenzyme and/or metal ions
apoenzyme (apoprotein)
protein part of enzyme
Some enzymes are modified covalently by __, ___ and other processes. These modifications are required to ___ enzyme activity.
phosphorylation, glycosylation
regulate
Alkaline Pase is found where?
in all tissues
high [c] in liver, bile duct, kidney, bone, placenta
Regulation of metabolic pathways involves 1+ of these (7) mechanisms:
1) amount of E present
2) [S]
3) reversible inhibition (by products or other compounds)
4) covalent modification
5) modulator protein binding
6) proteolytic cleavage
7) allosteric activation/inhibition
The regulatory mechanism used depends what on (2) things?
1) the function of the metabolic pathway where enzyme resides
2) purpose of regulation
Energy production pathways must be regulated by a mechanism that can do what?
respond quickly to ATP requirement
Storage pathways can be controlled by a mechanism that?
responds slowly to changing conditions
Allosteric Enzymes
enzymes that are regulated by binding of activators or inhibitors (allosteric effectors) to regulatory sites
Allosteric Enzyme - plot of reaction velocity vs. [S]
sigmodal
Non-regulatory Enzymes - plot of reaction velocity vs. [S]
hyperbolic
Allosteric effector molecules bind where?
bind to enzyme at site distinct and physically separate from S binding site
Allosteric Effectors affect?
overall S binding and/or reaction velocity
Homotropic effect
when S is effector molecule
Homotropic effects are observed when?
reaction of one S with E affects reaction of 2nd S with diff active site on multimeric protein.
The interaction between sub unites makes the binding of S ____ and results in what type of curve in plot of v vs. [S]?
cooperative
sigmoidal
Negative cooperativity
reaction of substrate with 1 active site makes it harder for another S to react at another active site
Positive cooperativity
reaction of substrate with 1 active site makes it easier for another S to react at another active site
Why can’t allosteric enzymes (with homotropic effector) be described by simple Michaelis-Menton Kinetics.
b/c affinity of E changes with [S]
How are allosteric enzymes with homotropic effectors characterized?
by [S] giving half-max rate [S]0.5 and Hill Coefficient, h
h values for:
1) (+) cooperativity
2) (-) negative cooperativity)
1) h>1
2) h<1
Feedback inhibition
a situation in which endpoint of pathway controls its rate of synthesis
usually takes place at 1st committed step, early step or branch point of pathway.
In feedback inhibition, __ usually inhibits or __ __ activates a regulatory enzyme in pathway.
endproduct
related metabolite
Feedback regulation often makes use of properties of? because?
allosteric enzymes b/c allosteric activators of inhibitors don’t need to resemble S or bind in active site
- also small changes in their [c] can have strong effect of velocity of rxn
In glycolysis, pyruvate kinase catalyzes which reaction?
last reaction between ADP & PEP
Pyruvate Kinase - regulation
allosterically regulated by ADP, ATP & alanine
feedforward activation by FBP
Glycolysis can function either ___ or ___ depending on (2)?
anaerobically & aerobically
availability of oxygen and ETC
Fate of pyruvate in cells with mitochondria and oxidative metabolism?
pyruvate converted completely to CO2 & H2O
- aerobic glycolysis
In RBCs that lack mitochondria & oxidative metabolism?
pyruvate reduced to lactate (3C hydroxyacid)
- anaerobic glycolysis
1 mol glucose –>—> 2 mols lactate
Ability of glycolysis to produce ATP under hypoxic conditions is especially important for?
skeletal muscles which can perform at high work output levels when O2 supply is limited
Other functions of glycolysis other than ATP production
in liver & adipose tissue - pyruvate is precursor for FA biosynthesis (regulated by glucagon & insulin)
- amino acids
- pentoses (5C sugars)
In yeast & other microorganisms, glycolysis represents..
1st stage in process of alcohol fermentation convert glucose to ethanol under anaerobic conditions
Alcohol Fermentation
pyruvate first decarboxylated to acetaldehyde by pyruvate decarboxylase
then acetaldehyde is reduced to ethanol by alcohol dehydrogenase
Pyruvate –> Acetaldehyde
2CH3COCOOH –> 2CH3CHO + 2CO2
acetaldehyde –> ethanol
2CH3CHO + 2NADH + 2H+ –> 2CH3CH2OH + 2NAD+
Overall run of Alcohol Fermentation
Glucose + 2Pi + 2ADP –> 2Ethanol + 2ATP + 2CO2
Iodoacetate inhibits?
G3P DH (converts G3P –> 1,3-diphosphoglycerate)
Fluoride inhibits?
enolase (converts 2-Phosphoglycerate to PEP)
Bisulfite (NaHSO4)
forms additional compound with acetaldehyde when it becomes unavailable for reaction with NADH in alcohol DH run
In most pathways of fuel oxidation, fats, carbs, proteins & ketone bodies are degraded to?
activated 2-C acetyl portion of acetyl-CoA
In the CAC, acetyl CoA is?
further oxidized to CO2 (oxidation occurs in 4 rxns that transfer e’s to e-accepting coenzymes NAD+ & FAD
4 reactions of the CAC involve oxidation (transferring e’s to NAD+ & FAD). The other reactions in CAC have what purpose?
rearrange electrons to facilitate e transfer to e-accepting coenzymes
Initially, what happens to acetyl-CoA in CAC?
combines with 4C intermediate OAA to form citrate (6C)
After formation of Citrate in CAC?
rearrangement of bonds in citrate followed by 2 oxidative decarboxylations (transfer e’s to NAD+ & release 2CO2)
After 2 oxidative decarbox. in CAC?
high E phosphate bond in GTP is generated from substrate-level phosphorylation
After substrate level phosphorylation in CAC?
2 e transfer reactions occur & OAA is regenerated
Overall process of CAC occurs with conservation of most of the E in chemical bonds of acetyl-CoA as..
3 NADH, 1FADH2, 1 GTP
Lactate is formed from..
degradation of carbohydrates via glycolysis
During hypoxic conditions (oxygen limited), cells turn towards?
anaerobic metabolism
In anaerobic metabolism, lactate DH…
converts pyruvate to lactate with concomitant NADH to NAD+. this increases [lactate] in blood
Since lactate is metabolized by ___ to produce __ via ____ , high blood [lactate] is usually found in people with?
liver
glucose
gluconeogenesis
poor liver function
One of the major products of amino acid metabolism is?
ammonia (NH3) which is highly toxic to higher organisms
In the liver, ammonia and CO2 are used to produce?
water-soluble form of nitrogen: urea via urea cycle
after urea is formed in urea cycle…
liver passes urea to blood which carries it to the kidney to be filtered out & excreted in urine
Why is increased [urea] in kidney an indicator of poor kidney?
one function of the kidney is to collect & excrete urea
Since urea is formed in the liver, low blood urea nitrogen is often the consequence of?
impaired liver function due to disease or infection (hepatitis)
(3) exothermic reactions that need to be bypassed for gluconeogenesis
1) hexokinase: glucose –> G6P (G6Pase)
2) PFK-1: F6P –>F16BP (F1,6-BPase-1)
3) pyruvate kinase: PEP –> pyruvate (pyruvate carboxylase, PEP carboxylase)
Gout
caused by excessive uric acid (ionized to urate in body) in the blood (final product of purine degradation) which causes inflammation of joints by precipitation of sodium urate crystals & can cause kidney stones (not very soluble)
Purpose of Allopurinol
prescribed for patients suffering from gout
Xanthase Oxidase inhibitor - (similar structure to xanthine)
- decreases [uric acid] - hypoxanthine & xanthine are soluble and can be excreted
energy from oxidation of fuels is converted to…. by process of..
high E phosphate bonds of ATP
oxidative phosphorylation
Most of the E from oxidation of fuels in TCA cycle & other oxidative pathways is conserved in the form of?
reduced e-accepting coenzymes, NADH & FADH2
The ETC does what?
oxidizes NADH & FADH2 & donates e’s to O2 which is reduced to H2O
E from reduction of O2 is utilized for the phosphorylation of ADP to ATP by ATP synthase
The entire ETC consists of several large __ ___ & 2 small independent components: __ & ___
protein complexes
ubiquinone (coenzyme Q) & cytochrome C
Electrons are conducted in a defined sequence from __ ___ through this system to __ and __ __ __ drives transport of __ from __ to ___
reduced coenzymes oxygen free energy change protons matrix to IMS
What provides a regulatory mechanism by which rate of ATP synthesis can control rate of e flow?
coupling of ATP synthesis to ETC through transmembrane proton gradient
As a consequence of ATP synthesis coupled to ETC…
rate of O2 consumption is coordinated with rate of ATP utilization
When ATP synthesis & e transport becomes ‘‘uncoupled” in brown adipose tissue, or in presence of chemical compounds…
E from ETC is converted to heat
Genetic diseases & other problems with ETC result in..
increased level of NADH which inhibited TCA & entry of pyruvate & FAs into cycle. Consequently, pyruvate is converted to lactate which appears in blood and FAs accumulate in tissues as TAGs.
Antimycin A inhibits?
Complex II
Malonate inhibits?
Complex II
Cyanide (CN-) & Carbon Monoxide (CO) inhibit?
Complex IV
(2) mechanism in humands for detox of xenobiotic materials & toxic metabolic waste
1) inactivation by reversibly binding to protein
2) chemical modification so it can be excreted (like ammonia –> urea)
Detox of Xenobiotic compounds is usually accomplished by.. or?
hydroxylation mediated by cyt P-450 enzymes or conjugation with sulcate or carbohyrate
Hydroxylation reaction consists of ?
NADPH, cyt reductase, cyt P-450 complex
cytochrome P-450 represents a class of..
heme containing monooxygenase induced by drugs such as coumarin
majority of cytochromes P450 are located in?
ER of liver
What determines the duration of drug action?
Rate of degradation of drugs
In Hydroxylation of drugs…
both NADH & NADPH donate reducing equivalents for reduction of cytochromes which in turn are oxidized by drugs in a series of hydrogenase reactions
Hydroxylation purpose
modifies functional groups which increases drugs solubility & loss of physiological activity
Conjugation of steroid hormones & phenolic drugs with sulfate & carbohydrate represents another …
significant mechanism for detox & removal of compounds
fate of Sulfate conjugated compounds
excreted directly in urine since they are more H2O soluble
fate of Carbohydrate conjugates
excreted into intestine lumen where hydrolysis of conjugates occurs & may result in some reabsorption of drug
Phase 1 - Hydroxylation: needs?
NADH & cyt P450 (microsomes)
Phase 2 - Conjugation of hydroxylated compounds with…
glucuronic acid, sulcate, acetate…
Phase 1 - Hydroxylation cycle
drug binds to P-450-Fe(3+), NADPH donates e’s through NADPH cyt450 reductase to ferric ion (becomes ferrous, Fe2+), oxygen enters & binds to drug, 1 oxygen of O2 goes to H2O, the other becomes the hydroxyl group (OH) attached to drug, Fe2+ becomes Ferric ion (Fe3+) again. Drug is then released.
Phase 2 - Conjugation pathway
UDP-glucose -(UDPG DH)-> UDP-glucuronate (reduction of 2NAD+ to 2NADH)
UDP-glucuronate donates glucuronate group to Drug-OH carboxylate group (- charge added) of glucuronate group on drug further increases solubility of hydroxylated compound
Cofactor function of B1 vitamin
aldehyde transfer
Cofactor function of B7 vitamin
carboxylation - helps aid transfer of CO2 groups
PDH is regulated by covalent mod & product inhibition. Identify products involved in inhibition & give name of covalent modification
Product Inhibition: acetyl-CoA, ATP, NADH
Covalent modification: phosphorylation/dephosphorylation
Monosaccharides other than glucose that can yield energy via glycolysis & identify reactions that are different from glucose involved in glycolysis
fructose/galactose/mannose
F6P cleaved by F1P aldolase forming glyceraldehyde & DHAP
glyceraldehyde is phosphorylated by ATP to form G3P
PDH plays significant role in regulation of glycolysis & oxidation of pyruvate. which mechanisms are responsible for this regulation?
product inhibition (ATP, NADH, acetyl CoA) & covalent modification (phos/dephos)
Glycolysis is connected to PPP through intermediates. Identify & show how intermediates are interconnected in these 2 pathways.
G6P, F6P, G3P are common to both pathways
Why is cells ability to regenerate NAD+ critical to glycolysis
coenzyme NAD+ is needed for G3P DH rxn where G3P becomes 1,3-BPG so without it, glycolysis can’t proceed
How many mols of ATP are generated in aerobic (1) and anaerobic (2) glycolysis
1) 32 mols of ATP
2) 2 mols of ATP
Which tissues/cells function under hypoxic conditions to produce lactate?
brain, erythrocytes (RBCs), skeletal muscle, renal medulla, retina
anapletoric reaction
enzyme catalyzed reaction that can replenish supply of metabolic intermediates of a metabolic pathway
example of anapletoric reaction
pyruvate carboxylase reaction
pyruvate –> OAA
(3) oxidative reactions along with enzymes in CAC in which NADH is produced
1) isocitrate –> α-KG + CO2 (isocitrate DH)
2) α-KG + CoA-SH –> Succinyl-CoA + CO2 (α-KG DH complex)
3) L-malate –> OAA (L-malate DH)
all with concomitant reduction of NAD+ to NADH
Cofactors of PDH reaction
1) TPP
2) FAD
3) Coenzyme A
4) NAD+
5) lipoate
Which cofactor of PDH is not classified as a vitamin?
lipase because it is a naturally occurring compound that is endogenously synthesized
Cataplerotic reaction
example?
reactions that remove intermediates from a pathway to
be used in other pathways
ex. α-KG + glutamine + NADPH + H+ –> 2 glutamate + NADP+
How is the energy of TCA cycle oxidations efficiently conserved?
conserved through reduction of 3 NAD+, 1 FAD, and 1 GDP (or ATP)
production 3 NADH, 1FADH2, 1 GTP
Explain why humans cannot convert fats to carbohydrates.
pyruvate kinase & pyruvate DH reactions are irreversible & exergonic. Acetate can’t serve as a starting material.
Substrates for gluconeogenesis in mammals? what role do fatty acids play in gluconeogenesis?
lactate, pyruvate, CAC intermediates, carbon skeletons of most aa’s.
FA breakdown provide ATP for gluconeogenesis
potential control points for gluconeogenesis & glycolysis
use different enzymes for 3 exergonic rxns.
hexokinase - g6pase
PFK- FBPase
Pyruvate Kinase- pyruvate carboxylase, PEP carboxylase
Outcome if individual has dietary thiamine deficiency?
pyruvate & α-KG DH are affected - impairment of pyruvate metabolism & lead to lactic acidosis causing neurologic disturbance
Why does alcohol consumption after strenuous exercise cause low blood glucose?
alcohol becomes oxidized by NAD+ and produces NADH. the lactate produced must be converted back to pyruvate but this rxn also produces NADH.
so lack of NAD+ inhibits gluconeogenesis
In amino acid metabolism in mammals, what are was finally broken down to?
amino group removed and formed urea. remaining carbon skeleton is broken down to CO2, h2o, glucose,acetyl-CoA or ketone body
the presence of transaminases in muscle & liver cells make them useful markers of tissue damage. Which transaminases are used for monitoring liver damage?
SGOT & SGPT
FEATURES shared by uncouplers?
hydrophobic & have dissociable H+
If a student takes a tablet and she starts to hyperventilate & become very hot, what is the most likely action of the tablet she has taken?
uncoupling ox. phos & ETC produces heat. Since there is no ATP synthesis, ADP accumulates, glycolysis & CAC increase to produce ATP & increase rate of ETC which increases rate of O2 consumption
In the inhibitor method for determining sequence of electron carriers, what is/are oxidized & what is/are reduced? Explain.
e carriers before step are reduced and e carriers after are oxidized
Why does iron deficiency result in fatigue?
decreased level of iron-containing cytochromes & Fe-S centres
- many E’s including cytochromes need iron as cofactor for catalysis
Although molecular oxygen doesn’t participate directly in any of the rxns of the CAC, why does the cycle operates only when O2 is present?
reduction of O2 reoxidizes NADH to NAD+ needed for glycolysis. If no O2 then NAD+ can’t be regenerated
What compounds are necessary for hydroxylation of coumarin to 7-hydroxycoumarin? functions of each.
NADPH - e donor
NADPH cyt450 reductase - facilitates e transfer to:
cytochrome P450 enzyme - makes drug more water soluble- binds it, chemically modifies it
Function of
1) UDGPA
2) microsomes
1) glucuronate donor
2) contain enzyme UDP-glucuronate transferase which catalyzes transfer of glucuronate to 7-hydroxycoumarin to form 7-OH coumaringlucuronide
