BIOCHEM BLOCK 3 REVIEW 2

Block 3 Review

• This is a focused view of a few major themes.
• Highlighted objectives are guaranteed to be covered.
• Additional details may be useful for successfully addressing objectives.

Exam Breakdown

• The exam has 57 questions in total.
• Below is a rough topic breakdown as 2 questions definitely cross topics and others may as well.
• Cholesterol Biosynthesis has 9 questions.
• Amino Acids and Nitrogen has 10 questions.
• Nucleotides and ATP has 9 questions.
• Digestion and Absorption has 8 questions.
• Proteoglycans and such has 8 questions.
• Molecular Processes has 6 questions.
• Neurochemistry has 7 questions.

Summary of Pathways for Exam

• Images will be provided for the Cholesterol Synthesis Pathway, Merged Amino Acid Degradation (deidentified), Urea Cycle, Purine Synthesis, and Pyrimidine Synthesis.
• Images will not be provided for HMG-CoA Reductase Regulation, Squalene Monooxygenase Regulation, Oxidative deamination, Dehydratase Activity, Absorptive/Fasting/Starvation States, Digestion and Absorption, Water and Electrolyte Movement, and Glycosylation (only major steps are an expectation).

Cholesterol Lecture Objectives

• Evaluate synthesis, uses, metabolism, and role in Cholesterol and Bile Salts in human health/disease
• Identify substrates, intermediate molecules, and metabolic products of cholesterol synthesis
• Explain the cross-over of Acetyl-CoA, HMG-CoA, and carbamoyl phosphate with other pathways in metabolism
• Assess the first committed step of Cholesterol synthesis regarding energy, substrates, products, consequences for cholesterol synthesis
• Identify conditions that would lead to cholesterol synthesis and those that would inhibit it
• Identify the role and regulation of HMGR, squalene synthase, squalene monooxygenase, oxidosqualene cyclase, and acyl-CoA:cholesterol acyltransferase (ACAT) in cholesterol synthesis and metabolism
• Assess the consequences for changes in the substrate availability and/or regulation of HMGR, squalene monooxygenase, oxidosqualene cyclase, and acyl-CoA:cholesterol acyltransferase (ACAT) in cholesterol synthesis and metabolism
• Identify the importance of carrier proteins and explain their role in cholesterol synthesis
• Identify the means through which cholesterol is packaged and transported through cells
• Compare and contrast mechanisms that increase and decrease the concentration of free cholesterol in terms of cholesterol homeostasis and assess for changes in cholesterol availability with changes in those mechanisms
• Describe the relationship between bile acids and bile salts based on pH
• Explain the role of bile acids/salts in digestion
• List the steps involved and the changes that occur as bile acids are utilized in digestion
• Assess the role of cholesterol and bile acids/salts in human diseases of the gallbladder and vasculature

HMGR Regulation

• Feedback inhibition occurs in HMGR regulation.
• Phosphorylation occurs in HMGR regulation.
• HMGR is inactive when phosphorylated by AMP-dependent kinase (AMPK).
• HMGR is active when not phosphorylated.
• HMGR regulation has a degradation rate.
• HMGR regulation contains sterol-sensing domain.
• High cholesterol causes binding and ubiquitylation of HMGR, leading to degradation.
• Hormone regulation occurs in HMGR regulation.
• Insulin and Triiodothyronine promote HMGR activity.
• Glucagon and Cortisol inhibits HMGR activity.
• Statins inhibit HMGR by binding the HMG-CoA binding site, therefore competitive inhibition.

Balancing Free Cholesterol

• Synthesis, breakdown of intracellular Cholesterol Esters by hydrolase activity, diet, and uptake of LDL via receptors/increase in LDL receptor concentration on surface of cell all increase cholesterol concentration
• Inhibition of synthesis, decrease in concentration of LDL receptors, esterification via ACAT, release to HDL, conversion to bile acids or steroid hormones, decreased HMG-CoA availability and/or decreased HMG-CoA Reductase (HMGR) activity, and high membrane concentration of cholesterol all decrease cholesterol concentration.

Amino Acid and Nitrogen Metabolism Objectives

• Summarize the mechanisms of protein degradation as part of metabolism.
• Emphasize the importance of protein turnover in proper development and maintenance of muscle in the adult.
• Compare and contrast amino acids in terms of synthesis and degradation.
• List the essential vs. nonessential amino acids.
• Explain the difference between essential and nonessential amino acids.
• Identify which amino acids are required in the diet and when there are additional dietary needs for certain amino acids.
• Connect an amino acid with the source material that generates it.
• Identify ketogenic and glucogenic amino acids.
• Summarize the processes of amino acid synthesis and degradation, linking enzymatic activity and necessary cofactors with the associated mechanism of degradation.
• Assess the causes or consequences for changes in the synthesis or degradation of amino acids, with emphasis on key enzymes involved in the digestion of amino acids.
• Summarize the Urea cycle and assess the consequences for changes in the urea cycle from the perspective of substrates, products, and particularly the enzymes involved.
• Link the changes in enzymes necessary for synthesis or degradation of amino acids or the process of nitrogen fixation (urea cycle) with the corresponding disorders.

Amino Acid Degradation

• Glucogenic amino acids can produce glucose in the liver.
• The Fumarate Group produces cytoplasmic fumarate.
• Ketogenic amino acids produce ketone bodies or acetyl CoA.

Phenylalanine Metabolism

• Dietary phenylalanine is processed into tyrosine via phenylalanine hydroxylase.
• Dihydropteridine reductase (DHPR) = dihydropteridine reductase.

Nucleotides and ATP Objectives

• Compare and contrast biosynthesis of purines and pyrimidines.
• Identify common elements of both pathways including energy needs.
• Summarize the enzymes involved.
• Explain the limitations and needs for purine and pyrimidine biosynthesis.
• Assess changes in synthesis based on cell type or enzyme activity.
• Assess purine and pyrimidine biosynthesis from the perspective of substrate availability and enzyme activity.
• Identify the rate-limiting steps of each process.
• Identify the likely causes or outcomes for enzyme deficiencies in purine and pyrimidine metabolism.
• Link the clinical symptoms/biochemical results with the corresponding disorder.
• Summarize the role of ATP and high energy bonds in bioenergetics/cellular metabolism.
• Determine when ATP synthesis would be induced vs. suppressed.

Purine vs Pyrimidine Biosynthesis

• Purine biosynthesis has a high energy cost.
• Sugars are added and then the base is added.
• The IMP precursor occurs in Purine Biosynthesis.
• Key intermediates in Purine Biosynthesis include ribose 5-phosphate, PRPP, GAR, AIR, SACAIR, and AICAR.
• Purine Biosynthesis generates AMP and GMP.
• Pyrimidine biosynthesis has a high energy cost.
• The bases are added and then the sugar is added.
• The UMP precursor occurs in Pyrimidine Biosynthesis.
• Key intermediates in Pyrimidine Biosynthesis include carbamoyl phosphate, dihydroorotate, and orotate.
• Pyrimidine Biosynthesis generates CTP and TMP to TTP.

Proteoglycans, Glycoproteins, Glycolipids, and Glycosylation Objectives

• Identify the types of glycosidic bonds and their relationship to chemical and molecular behavior of the molecule.
• Compare and contrast the structures of proteoglycans, glycoproteins, and glycolipids (Differentiate them based on description).
• Summarize the processes of N-linked glycosylation and O-linked glycosylation.
• Compare and contrast the location, enzymes, and amino acids involved in N-linked versus O-linked glycosylation.
• Assess the consequences of changes in the activity of enzymes associated with glycosylation and explain the role of glycosylation in human disease.
• Assess a case/condition and link symptoms with the most likely cause of the condition
• Determine the most appropriate testing to differentiate a condition from other conditions with similar symptomology

Biochemistry of Molecular Processes Objectives

• Summarize the processes associated with molecular genetics: replication, transcription, regulation of transcription, and translation.
• Explain steroid hormone-mediated gene regulation and the structures involved in the association of steroid receptors with DNA.
• Explain the process of charging t-RNA molecules and summarize the activities and critical features of aminoacyl tRNA synthetases.
• Identify the types of molecular analysis that can be performed and compare and contrast the analyses in diagnostics.
• Explain biomarkers and the role they can play in clinical medicine.
• Link changes in processes with the corresponding clinical features and identify the condition based on clinical phenotype.

Charging tRNA Molecules

• Aminoacyl-tRNA synthetases are a structurally diverse group.
• Discrimination of amino acids occurs in Aminoacyl-tRNA synthetases
• A mechanism involves the use of metal ions and ability to associate via charge.
• Chemical features of sidechains can be utilized in Aminoacyl-tRNA synthetases
• Aminoacyl-tRNA synthetases are a key player in Protein Sequence Fidelity.
• Aminoacyl-tRNA synthetases contain an editing mechanism to ensure proper attachment of corresponding amino acid.
• Editing can be pre- or post-transfer (essentially involving or immediately following the second step).
• Activation of the amino acid happens in two steps.
• Alpha-carboxylate oxygen of the amino acid to the alpha-phosphate group of the ATP, condensing into aminoacyl-adenylate (aa-AMP) occurs in two steps.
• Attachment to tRNA involves hydroxyl group of the adenine 76 nt attacks the carbonyl carbon of the adenylate, forming aminoacyl-tRNA and AMP

Digestion and Absorption Objectives

• Define key terms, including ingestion, digestion, absorption, maldigestion, malabsorption, and micronutrients.
• Identify and summarize the mechanisms involved in the digestion and absorption of nutrients.
• Identify the enzymes involved in digestion and absorption and explain their activation.
• Compare and contrast absorptive, fasting, and starved states and identify the changes in energy sources and metabolism for the liver, muscle, brain, adipose, RBC, and kidney.
• Assess the expected pattern of hormone expression and the metabolic behavior of the liver, muscle, brain, adipose, RBC, and kidney in fed, fasting, and starved states.
• Compare and contrast the digestion and absorption of carbohydrates, proteins, and fats.
• Assess the causes and consequences for altered digestion or absorption with an emphasis on enzymes involved.
• Link the altered enzyme with the observed change and identify the consequence if a change occurs.

Digestive Enzymes

• Most zymogen activation occurs in the gut lumen
• Most digestive enzymes are hydrolases
• Pepsinogen is activated by low pH to pepsin, which hydrolyzes peptides bonds adjacent to aromatic amino acids
• Prolipase is activated by binding with colipase to lipase
• Trypsinogen is activated by duodenal enteropeptidase hydrolyzing the N-terminal peptide to trypsin.
• Trypsin hydrolyzes peptide bonds adjacent to Arg and Lys.
• Chymotrypsinogen is activated by hydrolysis by enteropeptidase or trypsin to Chymotrypsin, which hydrolyzes peptide bonds adjacent to aromatic amino acids
• Proelastase is activated by hydrolysis by enteropeptidase or trypsin to elastase, which hydrolyzes peptide bonds adjacent to aliphatic amino acids
• Procarboxypeptidase A is activated by enteropeptidase or trypsin to Carboxypeptidase A, which hydrolyzes peptide bonds at carboxy terminal amino acids with aromatic or branched aliphatic side chains
• Procarboxypeptidase B is activated by enteropeptidase or trypsin to Carboxypeptidase B, which hydrolyzes peptide bonds at carboxy terminal amino acids with basic side chains

Basal Metabolism State

• Adapted from Lieberman and Peet, Basic Medical Biochemistry, 2018

Absorptive State

• Start of absorption until absorption is complete.
• Macronutrients from diet are oxidized to meet energy needs.
• Determinants of oxidation of fuel or storage for later use = endocrine hormones.
• Insulin increases.
• Glucagon is inhibited
• Adapted from Lieberman and Peet, Basic Medical Biochemistry, 2018

Short-Term Fasting

• Short-term fasting is similar to the basal state.
• It begins approximately 2 to 4 hours after a meal.
• Blood glucose declines to basal levels.
• Ends when blood glucose rises.
• Insulin levels reduce, and glucagon levels increase.
• Blood glucose is maintained by the liver.
• Adipose triacylglycerols are the major source of energy during fasting via lipolysis.
• Prolonged fasting causes changes to ensure that the body’s proteins are not consumed to the point of functional compromise.
• Adapted from Lieberman and Peet, Basic Medical Biochemistry, 2018.

Prolonged Fasting

• Entry into a starved state after 3 to 5 days of fasting.
• Fatty acid fuel is supplied by the breakdown of adipose tissue.
• Overall need for glucose is reduced.
• The brain utilizes increased blood ketone body levels, oxidizing them for energy to reduce the brain's glucose requirement.
• Glucose continues to be used, as it is needed for neurotransmitters.
• Red blood cells continue to use glucose as their sole energy source, due to a lack of mitochondria.
• Muscle decreases its use of ketone bodies, causing blood levels to rise and relying primarily on fatty acids for fuel.
• Liver stores of glycogen deplete after ~30 hours of fasting.
• Gluconeogenesis becomes the sole means of supplying blood glucose.
• Liver amino acid pool is the carbon source for glucose generation.
• The rate of gluconeogenesis is reduced, and urea production is decreased during prolonged fasting.
• The liver converts fatty acids into ketone bodies for use in other tissues.
• Adapted from Lieberman and Peet, Basic Medical Biochemistry, 2018.

Neurochemistry Objectives

• Compare and contrast the following neurotransmitters: Acetylcholine, Dopamine, Norepinephrine, Epinephrine, Serotonin, GABA, and substance P
• Summarize the process of neurotransmitter synthesis and degradation with emphasis on substrates, enzymes, metabolic products, and specific cell types associated with synthesis
• Explain the process of BH4 metabolism and identify the corresponding consequences of losses of proper BH4 metabolism
• Link/Identify substrates with crossover to other metabolic pathways including the pentose phosphate pathway (NADPH) and amino acids (glutamate, tyrosine, and tryptophan)
• Explain the limitation associated with inborn errors of metabolism in disorders of neurotransmitter synthesis and assess the consequences of pathogenic variation in neurotransmitter metabolism from the perspective of enzymes and cofactors

Neurotransmitters

• Neurotransmitters are an array of diverse chemicals
• They participate in cell-to-cell synaptic transmission within the CNS or PNS
• Each neuronal type produces a characteristic transmitter.
• Classes of Neurotransmitters include Acetylcholine (Ach), Monoamines (i.e. dopamine, norepinephrine, epinephrine, and serotonin), Neuropeptides (i.e. substance P), and simple amino acids (i.e. γ-aminobutyric acid (GABA), glutamic acid, aspartic acid, and glycine).
• Glucose is an important carbon source for the synthesis of both amino acids and neurotransmitters.
• Glutamate is a predominant excitatory neurotransmitter and a precursor of GABA.
• Sarnat HB and Flores-Sarnat L (2022) Neurology in Clinical Practice

Cofactors and Their Role in Neurotransmission

• Enzyme function is often dependent on cofactors.
• Enzyme function can be impaired or lost in the absence of cofactors.
• Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase.
• Loss of BH4 results in hyperphenylalaninemia.
• Cofactors are often produced by complex pathways, which is how regulation and important connection in the biochemical pathway occurs.
• BH4 metabolism can be correlated with the development of disorders.
• BH4 is oxidized in the hydroxylation reactions and thus must be regenerated.
• BH4 is regenerated by dihydropteridine reductase (DHPR) and pterin-4a-carbinolamine dehydratase (PCD).

Disorders of Neurotransmitter Synthesis

• Developmental disorders of neurotransmitter synthesis resulting from inborn metabolism errors are often incompatible with survival; particularly if impacting acetylcholine, monoamines, or an essential peptide.
• Defects in metabolic pathways associated with amino acids may cause intellectual disability, epilepsy, or neurological conditions, ex: Phenylketonuria

Don’t Forget the Cases!

• Cases can be used to define outcomes, enzymes involved, and changes in substrate and/or product.