quiz #7: murder that never was + DNA rep and repair

what is MMA?

genetic disease where the body cant properly break down certain fats and proteins because of a missing enzyme - methylmalonyl-CoA mutase (MUT)

what does MUT do? what happens when it doesn’t work?

MUT enzyme helps convert methylmalonyl-CoA into succinyl-CoA, which then feeds into the krebs cycle

when this enzyme doesn’t work (MMA), methylmalonyl-CoA builds up and gets converted into methylmalonic acid, which accumulates in the blood and becomes toxic

where is the major site of protein digestion?

small intestine

how are proteins digested?

1. proteins are broken down by proteolytic enzymes in the intestine to amino acids and oligopeptides

2. AA can be directly absorbed into the intestine, oligopeptides are broken down further by peptidases into amino acids

3. amino acids are then transported to the blood stream

how are amino acids stored

AA are not stored like fats or carbs, instead the body breaks up the amino group and the carbon skeleton

what is an ⍺-ketoacid?

COO- right next to =O

what are the ⍺-ketoacids in TCAC?

oxaloacetate and ⍺-ketoglutarate

what is an ⍺-amino acid?

NH2 group next to COOH

what is oxidative deamination?

the process by which the body removes an amino group from an amino acid to discard the nitrogen

1. gives the AA to an ⍺-ketoglutarate through an aminotransferasw, creating glutamate and a keto-acid (leftover carbon skeleton)

2. glutamate is processed by glutamate dehydrogenase where is loses its amino group and produces a free ammonia ionm regenerates ⍺-ketoglutarate so the cycle can start again

   1. energy is captured as NAD+ is reduced to NADH

is oxidative deamination catabolic or anabolic?

catabolic - breaks down amino acid

what are the ⍺-amino acids in the urea cycle?

1. citrulline

2. aspartate

3. arginosuccinate

4. arginine

5. ornithine

where does the urea cycle occur?

in the liver

what happens in the urea cycle?

the urea cycle gets ride of NH₄⁺ by turning it into urea, which is then excreted in the urine

1. ammonia and CO2 combine to form carbomoyl phosphate

2. carbamoyl phosphate joins ornithine to make citrulline

3. citrulline combines with aspartate to form arginosuccinate

4. arginosuccinate is broken into fumarate and arginine

5. arginine is split into urea and ornithine

   1. urea exits the cycle and is excreted

   2. ornithine is recycled

what does it mean to be glucogenic?

anything that can be used to produce glucose

what does it mean to be ketogenic?

anything that can be used to produce ketone bodies + fatty acids

• ketogenic carbon skeletons are converted to acetyl coA or acetoacetyl coA which can then be used to synthesize KB and FA

why can’t you use ketogenic things to form glucose?

you can’t go from acetyl-coA to pyurvate

what are 2 things you can do to verify GC peaks?

1. compare elution times - do each component separately

2. spiking in standard - add known component and see if a second spike is formed or if that spike grows

where does most amino acid degradation take place?

liver

what are the 2 sources of N in urea?

NH4+ and aspartate

what is the fate of carbon skeletons produced during AA degradation?

can be converted into TCAC intermediates, ketone bodies, or be used to synthesize glucose

what happened in ryan’s case?

• Patti was accused of poisoning her baby Ryan with antifreeze and ryan was put into foster care

• ryan died from another suspected poisoning after patti fed him a bottle and she was charged with 1st degree murder

  • ethylene glycol was thought to be found in his flood

• in prison, she gave birth to a second baby, DJ

• DJ started to have the same symptoms as ryan and was diagnosed with MMA

• the case against patti was revisited and tests confirmed that ryan also had mma

why would a MUT mutation cause elevated levels of propionic acid?

missing MUT → increased levels of methylmalonic coA

propionyl coA and propionic acid which are upstream fo the MUT reaction would increase as equilibrium shifts backwards

why can a vitamin B12 deficiency mimic a MUT deficiency?

the MUT enzyme requires a coenzyme derived from vitamin B12

• without this cofactor, the enzyme cant function even if its normal

• this causes a backup of methylmalonic-coA → MM acid build up just like in an actual MUT deficiency

how does a low energy charge affect oxidative deamination?

the reaction will be activated is AMP is high

• NADH generated by deamination can then be used by the ETC to produce ATP via oxidative phosphorylation to increase energy charge

why does the step with carbamoyl phosphate synthetase in the urea cycle need to be regulated?

this enzyme uses ammonia which is very toxic if it builds ip

regulated it controls how fast the urea cycle starts, making sure the body only removes nitrogen when needed

what happens to the ⍺-amino acid carbon skeletons after their ⍺-amino acid group is removed?

they get converted to 1 of 7 molecules:

1. acetyl-coA

2. acetoacetyl-coA

3. pyruvate

4. oxaloacetate

5. fumarate

6. succinyl-coA

7. ⍺-ketoglutarate

what is the link between TCAC and the urea cycle?

what were the results of laboratory A in ryans case?

they detected ethylene glycol at 180 mg/L and then at 911 mg/L after he was in foster care

how was propionic acid misidentified as ethylene glycol?

the first lab failed to verify the GC peak and assumed it was EG based solely on the elution time since the GC peak of propionic acid and ethylene glycol are about the same

the second lab spiked in ethylene glycol and saw 2 distinct peaks, meaning the original peak was not ethylene glycol. mass spec confirmed the unknown compound to be propionic acid

LO1: define a transamination reaction and identify alpha keto acids

transamination rxn: when an amino group is transferred from an AA to an alpha-keto acid (no free ammonia released)

alpha keto acid: molecules with C=O next to COOH

LO2: name the enzyme involved in oxidative deamination and how it is regulated in response to energy charge

glutamate dehydrogenase (removes the amino group from glutamate, releasing ammonia and producing alpha ketoglutarate)

• low energy charge activates GDH

• high energy charge inhibits GDH

LO3: state the purpose of the urea cycle and identify the alpha amino acids of the urea cycle

urea cycle safely removes toxic ammonia (NH4+) from the body by converting it into urea

• alpha AA: glutamate, aspartate

LO4: name the 7 molecules that are produced from the carbon skeletons of amino acids after deamination

1. pyruvate (G)

2. acetyl-coA (K)

3. acetoacetyl-coA (K)

4. alpha-ketoglutarate (G)

5. succinyl-coA (G)

6. fumarate (G)

7. oxaloacetate (G)

LO5: differentiate between ketogenic and glucogenic amino acids

GG: after breakdown they make molecules that can be turned into glucose and then feed into the krebs cycle

KG: after breakdown they make molecules that can be turned into ketone bodies or FA

LO6: state the function of the enzyme deficient in MMA and how that relates to lipid and AA metabolism

enzyme - methylmalonyl-coA mutase (MUT) which converted methylmalonyl-coA into succinyl coA which then enters the krebs cycle

FA and AA are broken down into MM-coA, which needs MUT to be process

without this MM-coA builds up and energy production from this is blocked

what are the purines and pyrimidines?

purines: AG (pure as gold) - 2 rings

pyrimidines: CUT (pyraminds cut) - 1 rings

how are nucleotides named?

1. [deoxy]

2. name of base

3. # of phosphates

4. phosphate

what are the base pairs?

A : T/U - 2H

C : G -3H

what are the major and minor grooves in DNA?

major groove: wider and deeper gap

minor groove: narrower and smaller gap

proteins often read the DNA by binding to the major groove

what does it mean that DNA is semiconservative?

the two complementary strands of DNA come apart and each serve as a template strand for a new DNA strand

how is a new nucleotide added onto a DNA strand?

the 3’ OH of the last nucleotide of the DNA strand attacks the alpha phosphate of the first phosphate group of the incoming nucleotide, kicking out a diphosphate → phosphodiester bond

catalyzed by DNA polymerase

ribose is the nucleophile, a-P is the electrophile

which end does DNA grow?

adds nucleotides onto the 3’ end

what is a nucleoside?

base + sugar (no phosphate)

where do you start numbering from on the ribose sugar?

start at the C to the right of O - OH should be at 3 (3’ OH)

what 2 types of non-covalent interactions contribute to the formation and stabilization of the DNA double helix?

VDW occurs between the stacked bases of one strand

HB occurs between the bases connecting two strands

what are the substrates of DNA polymerase?

primer, parent strand (template), a deoxynucleotidetriphosphate (dNTP - incoming nucleotide)

what atoms are directly involved in the reaction with DNA polymerase?

the 3’ OH of the last added nucleotide attacks the alpha phosphorus atom of the incoming nucleotide

what is lost from the incoming nucleotide by DNA polymerase?

2 PPi

what is the role of Mg²⁺ with DNA polymerase?

2 ions required

one stabilizes the O^- of the 3’ carbon on the primer strand, increasing its nucleophilicity leading to the attack

the other stabilizes the negative charges on the PPi leave group

(One of the magnesium ions is used to stabilize the negative charge of the 3' oxygen anion of the last nucleotide of the primer strand in order to increase its ability to attack as a nucleophile. The other magnesium ion is used to stabilize the negative charges on the β and γ phosphate groups that leave the molecule.)

why can DNA replication not occur in the 5’ → 3’ direction?

because then the reaction couldn’t be favored with the release of PPi, which is very favorable

the oxygen with the w P is a good LG to allow the bond to form between the 3’ O and the a-P of the incoming nucleotide

if it tried to proceed in the 3’ → 5’ direction?

• the 3’OH of the incoming nucleotide would have to attack the P group of the last added nucleotide

  • that is not possible as there is no good LG

  • if replication were to occur in the 3' to 5' direction, this energy would come from the 5' phosphate group already incorporated into the nucleotide, which is a less stable leaving group and thus less energetically favorable

where are the hydrogen bond donors and acceptors in base pairing?

what 2 reactions are necessary to convert C → T?

(1) a deamination reaction converts the amine group at the top to an keto group

(2) a methylation reaction adds a methyl group adjacent to the keto group

what are the sites in a DNA polymerase enzyme?

1. polymerase catalytic site (palm): where new nucleotides (dNTPs) are added to the growing DNA strand by a phosphodiester bond

2. 3’ exonuclease site: where proofreading happens

   1. removes incorrect bases in the 3’ → 5’ direction

are mistakes during DNA replication common or rare?

extremely rare1

what are the reasons why fidelity in DNA replication is high?

1. instability of mismatched pairs

   1. complementary BP have a much higher stability than mismatched pairs

2. configuration of the DNA polymerase active site

   1. DNA p is unlikely to catalyze bond formation between mismatched pairs

3. proofreading function of DNA polymerase

   1. DNA p can identify a mismatched nucleotide and remove it from the daughter strand

what causes DNA polymerase to pause or slow down during the process of DNA synthesis?

when a wrong NT is incorporated during DNA synthesis, the enzyme stalls due to the structural instability by the incorrect base pairing

• since the proper number of hydrogen bonds is required for a pairing to be stable, an error in this will cause the base pairing to be highly unstable, which DNA polymerase can detect due to the movement

the pause provides time for the incorrectly synthesized strand to be moved to the exonuclease active site of the enzyme, where wrong NT are removed

once the NT is removed, the strand is moved back to the polymerase active site and synthesis continues

what happens during the process of mismatch repair?

1. if the 3’ → 5’ exonuclease doesn’t catch it right away, MM repair steps in

2. the MutS protein complex finds the mismatch by recognizing the distortion caused by the wrong BP

3. MutL binds are helps coordinate the repair by finding which strand is new (not methylated)

4. MutH cuts out a section of the wrong DNA strand, including the mismatch

5. DNA polymerase fills the gap with the correct NT

MutS recognizes the incorrect BP and recruits MutL and MutH. MutL activates MutH which is an endonuclease that cleaves the newly synthesized DNA strand close to the mismatch

describe the MutS monomer domains?

• the clamp domains of each monomer initially recognize the mismatched DNA and have a positive charge on their surface, allowing for electrostatic attractions to form with the negatively charged DNA backbone

  • these interactions are sequence independent

  • the DNA molecule can now make contact with the mismatch-recognitition monomer of MutS through its bases and the non-mismatch binding monomer of MutS through its backbone

compare the 2 MutS monomers?

mismatch recognition monomer:

• binds the major groove

• base-specific interactions

• uses the mismatch-binding domain

non-mismatch binding monomer:

• binds the minor groove

• electrostatic interactions

• uses the clamp domain