Biochem Finals Post 2024-2025 PDF
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Uploaded by ConsummateLilac
2025
Heshynee Mae Aiko T. Tagaro
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Summary
This document is a past biochemistry finals post-laboratory discussion covering several topics including DNA extraction from fruit, mutation, hormones, cholesterol biosynthesis, and amino acid degradation. It includes detailed explanations and examples in each section. This document is suitable for biochemistry undergraduate students preparing for an exam.
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DNA Extraction and Mutation Heshynee Mae Aiko T. Tagaro, RPh, MA Bio Finals Post-Laboratory Discussion 1st semester AY 2024-2025 DNA extraction from fruit over-ripe banana is best since the cell walls are already...
DNA Extraction and Mutation Heshynee Mae Aiko T. Tagaro, RPh, MA Bio Finals Post-Laboratory Discussion 1st semester AY 2024-2025 DNA extraction from fruit over-ripe banana is best since the cell walls are already decomposing physical mashing continues to break up the cell walls DNA extraction from fruit The salt solution helps the DNA to aggregate (clump together). DNA extraction from fruit dissolves the lipids in the cell and nuclear membranes releases DNA into the salt solution DNA extraction from fruit Pour mash through filter into a beaker DNA extraction from fruit Pour about 5 ml of filtrate into a test tube DNA extraction from fruit Slowly pour an EQUAL volume of cold ethanol down the side of tube to form a layer on top of the fruit fluid (to form a separate layer on top of the fruit solution). Do not mix the alcohol and banana solution. Ice-cold 100% ethanol works best DNA extraction from fruit Spool the DNA: use a glass rod to gently swirl at the interface of the two solutions. the interface is where the two solutions meet DNA is not soluble in alcohol bubbles may form around a wooly substance (this is the DNA) DNA extraction from fruit transfer the DNA in an empty tube and start identification test Dische Diphenylamine Test For DNA Acidic conditions convert deoxyribose to a molecule that binds with diphenylamine to form a blue complex. The Dische’s Test will detect the deoxyribose of DNA and will not interact with the ribose in RNA. A clear tube indicates no nucleic acids. A blue color indicates the presence of DNA. A greenish color indicates the presence of RNA. Mutation An error in base sequence reproduced during DNA replication. Errors in genetic information is passed on during transcription The altered information can cause changes in amino acid sequence during protein synthesis and thereby alter protein function. Mutation is change of base pairs. – It can be either of the two: Transitional – NB replaces the same type of NB Transversional – NB replaces different type of NB Results of mutation Silent mutation: There is a change but the protein still codes for the same amino acid. [example: UCA (serine) to UCU (serine)] Missense mutation: A different codon is produced therefore it codes for a different amino acid [example:UCA (serine) to CCA (proline)] Nonsense mutation: A stop codon is produced thereby also stopping protein synthesis [example: UCA to UAA (stop)] Hormones LIPID-DERIVED HORMONES derived from cholesterol Chemically, these hormones are usually ketones or alcohols; their chemical names will end in “-ol” for alcohols or “-one” for ketones. Steroid hormones are insoluble in water, and they are transported by transport proteins in blood. – As a result, they remain in circulation longer than peptide hormones. For example, cortisol has a half-life of 60 to 90 minutes, while epinephrine, an amino acid derived-hormone, has a half-life of approximately one minute. AMINO ACID-DERIVED HORMONES relatively small molecules that are derived from the amino acids tyrosine and tryptophan If a hormone is amino acid- derived, its chemical name will end in “-ine”. PEPTIDE HORMONES include molecules that are short polypeptide chains, such as antidiuretic hormone and oxytocin includes small proteins, like growth hormones and large glycoproteins such as follicle-stimulating hormone Amino acid-derived and polypeptide hormones are water-soluble and insoluble in lipids. These hormones cannot pass through plasma membranes of cells; therefore, their receptors are found on the surface of the target cells. 17 18 19 20 Cholesterol biosynthesis The process of cholesterol synthesis can be considered to be composed of five major steps where the reactions that culminate in the synthesis of isopentenyl pyrophosphate (IPP), and its isomeric form, dimethylallyl pyrophosphate (DMAPP), are commonly referred to as the mevalonate pathway: 1. Acetyl-CoAs are converted to 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA) 2. HMG-CoA is converted to mevalonate 3. Mevalonate is converted to the isoprene based molecule, isopentenyl pyrophosphate (IPP) 4. IPP molecules are converted to squalene 5. Squalene is converted to cholesterol acetyl CoA is the starting compound and NADPH is the coenzyme rate limiting enzyme: (1) HMG-CoA reductase and (2) SQLE – A rate-limiting enzyme is a key enzyme of which the activity determines the overall rate of a metabolic pathway. Intermediates in the mevalonate pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of heme-α. 3-hydroxy-3-methylglutaryl coenzyme A HMGCR: HMG-CoA reductase FDPS: farnesyl diphosphate synthase FDFT1: farnesyl-diphosphate isopentenyl pyrophosphate farnesyltransferase 1 (more commonly called squalene synthase) farnesyl pyrophosphate SQLE: squalene epoxidase (also called squalene monooxygenase Kandutsch-Russell pathway. Fates of the Carbon Skeletons of Amino Acids Pyruvate as an Entry Point into Metabolism Pyruvate is the point of entry for alanine, serine, cysteine, glycine, threonine, and tryptophan. The transamination of alanine directly yields pyruvate. The deamination of serine to pyruvate by serine dehydratase. Cysteine can be converted into pyruvate. Glycine can be converted into serine by enzymatic addition of a hydroxymethyl group or it can be cleaved to give CO2, NH4+, and an activated one-carbon unit. Threonine can give rise to pyruvate through the intermediate aminoacetone. Three carbon atoms of tryptophan can emerge in alanine, which can be converted into pyruvate. 27 Oxaloacetate as an Entry Point into Metabolism Aspartate and asparagine are converted into oxaloacetate, a citric acid cycle intermediate. – Aspartate, a four-carbon amino acid, is directly transaminated transaminated to oxaloacetate. – Aspartate can also be converted into fumarate by the urea cycle – Asparagine is hydrolyzed by asparaginase to NH4+ and aspartate, which is then transaminated. Alpha-Ketoglutarate as an Entry Point into Metabolism The carbon skeletons of several five- carbon amino acids enter the citric acid cycle at α-ketoglutarate. These amino acids are first converted into glutamate, which is then oxidatively deaminated by glutamate dehydrogenase to yield α- ketoglutarate. Histidine Degradation Histidine is converted into 4-imidazolone 5- propionate. The amide bond in the ring of this intermediate is hydrolyzed to the N-formimino derivative of glutamate, which is then converted into glutamate by transfer of its formimino group to tetrahydrofolate, a carrier of activated one- carbon units. Proline and arginine are each converted into glutamate γ-semialdehyde, which is then oxidized to glutamate. Glutamine is hydrolyzed to glutamate and NH4+ by glutaminase. Succinyl Coenzyme A Is a Point of Entry for Several Nonpolar Amino Acids Succinyl CoA is a point of entry for some of the carbon atoms of methionine, isoleucine, and valine. Propionyl CoA and methylmalonyl CoA are intermediates in the breakdown of these three nonpolar amino acids This pathway from propionyl CoA to succinyl CoA is also used in the oxidation of fatty acids that have an odd number of carbon atoms. BCAA's yield acetyl CoA, Acetoacetate or Propionyl CoA Leucine is transaminated to the corresponding α-ketoacid, α-ketoisocaproate. This α-ketoacid is oxidatively decarboxylated to isovaleryl CoA by the branched-chain α-ketoacid dehydrogenase complex. 36 The isovaleryl CoA derived from leucine is dehydrogenated to yield β-methylcrotonyl CoA. This oxidation is catalyzed by isovaleryl CoA dehydrogenase. β-Methylglutaconyl CoA is then formed by the carboxylation of β-methylcrotonyl CoA at the expense of the hydrolysis of a molecule of ATP 37 β-Methylglutaconyl CoA is then hydrated to form 3-hydroxy-3- methylglutaryl CoA (HMG-CoA), which is cleaved into acetyl CoA and acetoacetate. 38 The degradative pathways of valine and isoleucine resemble that of leucine. Isoleucine yields acetyl CoA and propionyl CoA, whereas valine yields CO2 and propionyl CoA. Degradation of Aromatic Amino Acids The degradation of phenylalanine begins with its hydroxylation to tyrosine, a reaction catalyzed by phenylalanine hydroxylase. This enzyme is a monooxygenase because one atom of O2 appears in the product and the other in H2O. The reductant here is tetrahydrobiopterin, an electron carrier derived from the cofactor biopterin. The next step is the transamination of tyrosine to p-hydroxyphenylpyruvate. This α-ketoacid then reacts with O2 to form homogentisate. The enzyme catalyzing this complex reaction, p- hydroxyphenylpyruvate hydroxylase, is a dioxygenase because both atoms of O2 become incorporated into the product, one on the ring and one in the carboxyl group. The aromatic ring of homogentisate is then cleaved by O2, which yields 4- maleylacetoacetate. This reaction is catalyzed by homogentisate oxidase, another dioxygenase. 4-Maleylacetoacetate is then isomerized to 4-fumarylacetoacetate by an 4-maleylacetoacetate isomerase that uses glutathione as a cofactor. Finally, 4-fumarylacetoacetate is hydrolyzed to fumarate and acetoacetate. Tryptophan Degradation Tryptophan degradation requires several oxygenases. Tryptophan 2,3-dioxygenase cleaves the pyrrole ring, and kynureinine 3-monooxygenase hydroxylates the remaining benzene ring. Alanine is removed and the 3-hydroxyanthranilic acid is cleaved with another dioxygenase and subsequently processed to acetoacetyl CoA. Nearly all cleavages of aromatic rings in biological systems are catalyzed by dioxygenases. – The active sites of these enzymes contain iron.