Molecular Biology Lab Exam 2 Study Guide

POLG function

The role of the POLG gene in mitochondrial diseases.

MCHS

Myocerebrohepatopathy Spectrum, a mitochondrial disease with neonatal onset involving liver failure, lactic acidosis, and encephalopathy.

AHS

Alpers-Huttenlocher Syndrome, an infant-onset mitochondrial disease characterized by progressive cerebral degeneration, liver failure, and intractable seizures.

MNGIE

Mitochondrial Neurogastrointestinal Encephalopathy, a childhood-onset mitochondrial disease associated with GI dysmotility, leukoencephalopathy, and cachexia.

MIRAS

Mitochondrial Recessive Ataxia Syndrome, an adolescence-onset mitochondrial disease with ataxia, seizures, and white matter changes.

SCAE

Spinocerebellar Ataxia with Epilepsy, an adolescence-onset mitochondrial disease that overlaps with MIRAS and includes generalized seizures.

MEMSA

Myoclonic Epilepsy Myopathy Sensory Ataxia, an adolescence-onset mitochondrial disease characterized by myopathy, epilepsy, and ataxia.

MELAS

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes, a classic mtDNA mutation syndrome with maternal inheritance.

PEO

Progressive External Ophthalmoplegia, a mitochondrial disease with eyelid droop and ophthalmoplegia, often involving POLG and other genes.

ANS

Ataxia Neuropathy Spectrum, a mitochondrial disease in adults characterized by ataxia, peripheral neuropathy, and possibly PEO or seizures.

SANDO

Sensory Ataxic Neuropathy, Dysarthria, and Ophthalmoparesis, an adult-onset mitochondrial disease that falls within the ANS-PEO-MIRAS continuum.

Hepatocerebral MDDS

Mitochondrial DNA depletion syndromes involving epilepsy and seizures.

ATP consumption in brain

The brain, which is ~2% of body weight, consumes ~20% of total body ATP.

OXPHOS

Oxidative phosphorylation, a process by which neurons generate ATP from mitochondria.

Cerebellum and hippocampus

Regions of the brain important in motor control and seizure thresholds, vulnerable to mitochondrial dysfunction.

Molecular epistasis

The interaction of different genes where one gene's effect is modified by another gene, particularly in the context of haploinsufficiency.

Known MDDS loci

Genes associated with mitochondrial DNA depletion syndromes, including TYMP, TK2, RRM2B, SUCLG1, SUCLG2, DGUOK, MPV17, POLG2, and TWNK.

Purine anabolic pathways

Biochemical pathways that synthesize purines, facilitated by the purinosome.

Pyrimidine biosynthesis

The biochemical process of synthesizing pyrimidine nucleotides.

One-carbon folate shuttling

The transport of one-carbon units in metabolic processes, involving SHMT, TS, and DHFR.

mtDNA replisome

The complex of proteins involved in the replication of mitochondrial DNA, including TFAM, POLRMT, POLG2, TWNK, SSBP1, FEN1, RNASEH1, and LIG3.

Heme biosynthesis pathway

A 10-step biochemical pathway for synthesizing heme, with specific steps occurring on the cristae membrane.

TIM/TOM complex

Translocase of the inner membrane (TIM) and translocase of the outer membrane (TOM), involved in trafficking mitochondrial-localization sequences across mitochondrial membranes.

TIM/TOM complex

Complex for trafficking mitochondrial-localization sequences (MLS aka presequence) protein cargo across the inner membrane (TIM) or outer membrane (TOM) of the mitochondria.

TYMP

Known MDDS locus associated with neurogastrointestinal MDDS.

TK2

Known MDDS locus associated with myopathic MDDS.

RRM2B

Known MDDS locus associated with encephalomyopathic MDDS.

SUCLG1

Known MDDS locus associated with encephalomyopathic MDDS.

SUCLG2

Known MDDS locus associated with encephalomyopathic MDDS.

DGUOK

Known MDDS locus associated with hepatocerebral MDDS.

MPV17

Known MDDS locus associated with hepatocerebral MDDS.

POLG2

Known MDDS locus associated with hepatocerebral MDDS.

TWNK

Known MDDS locus associated with hepatocerebral MDDS.

purinosome

Loss of purinosome function will likely be epistatic to POLG haploinsufficiency.

purine biosynthesis

Involves 10 distinct enzymatic steps from PRPP to IMP.

PRPP

Supplies the 5' phosphate group as well as the pentose sugar (ribose) in purine biosynthesis.

GAR synthetase

Enzyme in step 2 of purine biosynthesis that incorporates a glycine amino acid into the developing imidazole ring.

GAR transformylase

Enzyme in step 3 of purine biosynthesis that uses GAR and 10-formyl-THF to produce FGAR and THF.

10-formyl-THF

Part of the 1C shuttle system, derived from dietary vitamin B9 (folate), essential for THF synthesis.

FGAR amidotransferase

Enzyme in step 4 of purine biosynthesis that transfers a nitrogen from Gln to form part of the future pyrimidine ring.

AIR synthetase

Enzyme in step 5 of purine biosynthesis that circularizes the imidazole ring of the future purine base.

AIR carboxylase

Enzyme in step 6 of purine biosynthesis that fixes carbon dioxide as a carboxylic group in the future pyrimidine ring.

N5-CAIR synthetase

In plants, fungi, and bacteria, an enzyme that converts AIR to N5-CAIR using ATP and bicarbonate.

N5-CAIR mutase

In plants, fungi, and bacteria, an enzyme that converts N5-CAIR to CAIR.

CAIR

A compound involved in de novo purine biosynthesis.

SAICAR synthetase

An enzyme that catalyzes the conversion of CAIR, Asp, and ATP into SAICAR, ADP, and Pi.

Step 7 of de novo purine biosynthesis

The step where CAIR is converted to SAICAR.

Step 8 of de novo purine biosynthesis

The step where SAICAR is converted to AICAR and fumarate.

Adenylosuccinate lyase

An enzyme that catalyzes the conversion of SAICAR to AICAR and fumarate.

Fumarate

A compound that is essentially aspartic acid that has been deaminated.

Bifunctional enzyme

An enzyme that has two distinct functional activities, such as the adenylosuccinate lyase.

Step 9 of de novo purine biosynthesis

The step where AICAR is converted to FAICAR and THF.

AICAR transformylase

An enzyme that catalyzes the conversion of AICAR and 10-formyl-THF into FAICAR and THF.

Transformylase reactions

Reactions that utilize 1C shuttles for the transfer of carbon units.

Step 10 of de novo purine biosynthesis

The step where FAICAR is converted to IMP and H2O.

IMP cyclohydrolase

An enzyme that catalyzes the conversion of FAICAR to IMP.

IMP

Inosine monophosphate, an intermediate in purine biosynthesis.

Overall purine biosynthesis pathway

A pathway consisting of 10 catalytic steps that is very endergonic and requires various amino acids and cofactors.

Folate shuttles

1C carriers required in the purine biosynthesis pathway.

Purinosome

A hexameric complex in humans that contains 10 active sites for the conversion of PRPP to IMP.

PPAT

An enzyme that contains E1 in the purinosome.

GART

A trifunctional enzyme in the purinosome that contains E2, E3, and E5.

PFAS

An enzyme in the purinosome that contains E4.

PAICS

A bifunctional enzyme in the purinosome that contains E6 and E7.

ADSL

An enzyme in the purinosome that contains E8.

ATIC

A bifunctional enzyme in the purinosome that contains E9 and E10.

Intermediate channeling

A process in the purinosome that exploits Le Chatelier's Principle and protects metabolites from unwanted reactions.

SHMT complex

A complex that performs a key reaction for the purinosome involving L-Ser and THF.

C1-THF-synthase complex

A complex that performs two key reactions for the purinosome involving 5,10-methylene-THF.

IMP to XMP to GMP pathway

The pathway where IMP is converted to XMP and then to GMP.

G-1 enzyme

The enzyme that catalyzes the reaction of IMP to XMP.

G-2 enzyme

The enzyme that catalyzes the reaction of XMP to GMP.

XMP

xanthosine monophosphate

NADH

Nicotinamide adenine dinucleotide (reduced form)

H+

Proton ion

G-1 enzyme

Enzyme that converts IMP to GMP, inhibited by high concentrations of GMP

G-2 enzyme

GMP synthetase reaction

Input for G-2 enzyme

XMP, Gln, ATP

Output for G-2 enzyme

GMP, Glu, ADP, Pi

A-1 enzyme

adenylosuccinate synthetase

Input for A-1 enzyme

IMP, Asp, GTP

Output for A-1 enzyme

adenylosuccinate, GDP, Pi

Negative allosterism on A-1 enzyme

AMP

A-2 enzyme

adenylosuccinate lyase

Input for A-2 enzyme

adenylosuccinate

Output for A-2 enzyme

AMP, fumarate

GTP production from GMP

GMP + ATP -(guanylate kinase)-> GDP + ADP

Second reaction for GTP production

GDP + ATP -(NDP kinase)-> GTP + ADP

ATP hydrolysis for GTP production

Need to hydrolyze 2 ATP molecules (1 ATP hydrolysis per step) to make one GTP

GTP characteristics

GTP belongs to the RNA world (has ribose not deoxyribose moiety)

AMP to ADP reaction

AMP + ATP -(adenylate kinase)-> 2 ADP

Pyrimidine biosynthesis

Occurs at the free nucleobase level

Carbamoyl phosphate source

Source of 2 atoms (1 N, 1 C) for pyrimidine biosynthesis

Aspartic acid source

Source of 4 atoms (1 N, 3 C) for pyrimidine biosynthesis

Step 1 of de novo pyrimidine biosynthesis

Enzyme: carbamoyl phosphate synthetase, Input: Gln, HCO3-, H2O, 2 ATP, Output: Carbamoyl phosphate, Glu, 2 ADP, Pi

Step 2 of de novo pyrimidine biosynthesis

Enzyme: aspartate transcarbamoylase, Input: Carbamoyl phosphate, Asp, Output: Carbamoyl aspartate, Pi

Positive allosteric modulator of aspartate transcarbamoylase

ATP

Negative allosteric modulator of aspartate transcarbamoylase

CTP

Step 3 of de novo pyrimidine biosynthesis

Enzyme: dihydroorotase, Input: carbamoyl aspartate, Output: dihydroorotate, H2O

Step 4 of de novo pyrimidine biosynthesis

Enzyme: dihydroorotate dehydrogenase, Input: dihydroorotate, coenzymeQ, Output: orotate, CoQH2