CHEM113 Lecture Notes PDF

CHEM113 – LECTURE COVERAGE 2. Type of reaction catalyzed by an enzyme is often used as a prefix 1. Enz...

CHEM113 – LECTURE COVERAGE 2. Type of reaction catalyzed by an enzyme is often used as a prefix 1. Enzymes and Vitamins  E.g., Oxidase - catalyzes an oxidation reaction, 2. Nucleic Acids  E.g., Hydrolase - catalyzes a hydrolysis reaction 3. Digestion 3. Identity of substrate is often used in addition to the type of 4. Bioenergy Production reaction  E.g. Glucose oxidase, pyruvate carboxylase, and succinate ENZYMES AND VITAMINS dehydrogenase - Enzymes are catalysts and are not consumed in the reactions - Enzymes are proteins that act as a catalyst for biochemical Six Major Classes reactions - Enzymes are grouped into six major classes based on the types - The human body has 1000s of enzymes of reactions they catalyze - Enzymes are the most effective catalysts known Class Reaction Catalyzed - Most enzymes are globular proteins 1. Oxidoreductases Oxidation-reductions - A few enzymes are now known to be ribonucleic acids (RNA) 2. Transferases Functional group transfer reactions - Enzymes undergo all the reactions of proteins including 3. Hydrolases Hydrolysis reactions denaturation 4. Lyases Reactions involving addition or removal - Enzyme activity is dramatically affected by: of groups form double bonds  Alterations in pH 5. Isomerase Isomerisation reactions  Temperature 6. Ligases Reactions involving bond formation  Other protein denaturants coupled with ATP hydrolysis Simple and Conjugated Enzymes Oxidoreductase  Simple enzyme: composed only of protein (amino acid chains) - catalyzes an oxidation–reduction reaction:  Conjugated enzyme: Has a nonprotein part in addition to a  Oxidation and reduction reactions are always linked to one protein part. another o Apoenzyme: Protein part of a conjugated enzyme.  An oxidoreductase requires a coenzyme that is either o A cofactor: Nonprotein part of a conjugated enzyme. oxidized or reduced as the substrate in the reaction. o A holoenzyme is the biochemically active conjugated  E.g., Lactate dehydrogenase is an oxidoreductase and the enzyme reaction catalyzed is shown below o Apoenzyme + cofactor = holoenzyme (conjugated enzyme) Transferase - an enzyme that catalyzes the transfer of a functional group from Cofactors one molecule to another - important for the chemically reactive enzymes - Two major subtypes: - small organic molecules or Inorganic ions - Organic molecule cofactors: also called as co-enzymes or co-  Transaminases - catalyze transfer of an amino group to a substrates substrate - Co-enzymes/co-substrates are derived from dietary vitamins  Kinases - catalyze transfer of a phosphate group from - Inorganic ion cofactors adenosine triphosphate (ATP) to a substrate - Typical metal ion cofactors - Zn2+, Mg2+, Mn2+, and Fe2+ Hydrolase - Nonmetallic ion cofactor - Cl- - A hydrolase is an enzyme that catalyzes a hydrolysis reaction - Inorganic ion cofactors derived from dietary minerals - The reaction involves addition of a water molecule to a bond to cause bond breakage Nomenclature and Classification of Enzymes - Hydrolysis reactions are central to the process of digestion: - Nomenclature: Most commonly named with reference to their  Carbohydrase’s hydrolyze glycosidic bonds in oligo- and function polysaccharides (see reaction below)  Type of reaction catalyzed  Proteases effect the breaking of peptide linkages in proteins,  Identity of the substrate  Lipases effect the breaking of ester linkages in - A substrate is the reactant in an enzyme-catalyzed reaction: triacylglycerols  The substrate is the substance upon which the enzyme Lyase “acts.” - an enzyme that catalyzes the addition of a group to a double  E.g., In the fermentation process sugar to be converted to bond or the removal of a group to form a double bond in a CO2, therefore in this reaction sugar is the substrate manner that does not involve hydrolysis or oxidation  Dehydratase: effects the removal of the components of Three Important Aspects of the Naming Process water from a double bond 1. Suffix -ase identifies it as an enzyme  Hydratase: effects the addition of the components of water  E.g., urease, sucrase, and lipase are all enzyme to a double bond designations Isomerase and Ligase  Exception: The suffix -in is still found in the names of some - An isomerase is an enzyme that catalyzes the isomerization digestive enzymes, E.g., trypsin, chymotrypsin, and pepsin (rearrangement of atoms) reactions. Aki | 1  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM - A ligase is an enzyme that catalyzes the formation of a bond Factors that affect Enzyme Activity between two molecules involving ATP hydrolysis: Temperature - ATP hydrolysis is required because such reactions are - Higher temperature results in higher kinetic energy which energetically unfavorable causes an increase in number of reactant collisions, therefore - Require the simultaneous input of energy obtained by a there is higher activity. hydrolysis of ATP to ADP - Optimum temperature: Temperature at which the rate of enzyme catalyzed reaction is maximum Models of Enzyme Action - Optimum temperature for human enzymes is 37ºC (body Enzyme Active Site temperature) - The active site: Relatively small part of an enzyme’s structure - Increased temperature leads to decreased enzyme activity that is actually involved in catalysis: pH  Place where substrate binds to enzyme - pH changes affect enzyme activity  Formed due to folding and bending of the protein. - Drastic changes in pH can result in denaturation of proteins  Usually a “crevice like” location in the enzyme - Optimum pH: pH at which enzyme has maximum activity  Some enzymes have more than one active site - Most enzymes have optimal activity in the pH range of 7.0 - 7.5 Enzyme Substrate Complex - Exception: Digestive enzymes - Needed for the activity of enzyme - Pepsin: Optimum pH = 2.0 - Intermediate reaction species formed when substrate binds with - Trypsin: Optimum pH = 8.0 the active site Substrate Concentration - Orientation and proximity is favorable and reaction is fast - Substrate Concentration: At a constant enzyme concentration, Two Models for Substrate Binding to Enzyme the enzyme activity increases with increased substrate Lock-and-Key model: concentration. - Enzyme has a pre-determined shape for the active site - Substrate saturation: the concentration at which it reaches its - Only substrate of specific shape can bind with active site maximum rate and all of the active sites are full Induced Fit Model: - Turnover Number: Number of substrate molecules converted to - Substrate contact with enzyme will change the shape of the product per second per enzyme molecule under conditions of active site optimum temperature and pH - Allows small change in space to accommodate substrate Enzyme Concentration (e.g., how a hand fit into a glove) - Enzymes are not consumed in the reactions they catalyze Forces That Determine Substrate Binding - At a constant substrate concentration, enzyme activity increases  H-bonding with increase in enzyme concentration  Hydrophobic interactions - The greater the enzyme concentration, the greater the reaction  Electrostatic interactions rate. Enzymes Inhibition Enzyme specificity - Enzyme Inhibitor: a substance that slows down or stops the Absolute Specificity - enzyme will catalyze a particular reaction for only one substrate normal catalytic function of an enzyme by binding to it. - Competitive Inhibitors: Compete with the substrate for the same - This is most restrictive of all specificities (not common) active site - E.g., Urease is an enzyme with absolute specificity Stereochemical Specificity  Will have similar charge & shape - An enzyme can distinguish between stereoisomers.  Noncompetitive Inhibitors: Do not compete with the - Chirality is inherent in an active site (amino acids are chiral substrate for the same active site compounds)  Binds to the enzyme at a location other than active site - L-Amino-acid oxidase - catalyzes reactions of L-amino acids Reversible Competitive Inhibition but not of D-amino acids. - A competitive enzyme inhibitor: resembles an enzyme Group Specificity substrate in shape and charge - Involves structurally similar compounds that have the same - Binds reversibly to an enzyme active site and the inhibitor functional groups. remains unchanged (no reaction occurs) - E.g., Carboxypeptidase: Cleaves amino acids one at a time from - The enzyme - inhibitor complex formation is via weak the carboxyl end of the peptide chain interactions (hydrogen bonds, etc.). Linkage Specificity - Competitive inhibition can be reduced by simply - Involves a particular type of bond irrespective of the structural increasing the concentration of the substrate. features in the vicinity of the bond Reversible Noncompetitive Inhibition - Considered most general of enzyme specificities - A noncompetitive enzyme inhibitor decreases enzyme - E.g., Phosphatases: Hydrolyze phosphate–ester bonds in all activity by binding to a site on an enzyme other than the types of phosphate esters active site. - Causes a change in the structure of the enzyme and prevents enzyme activity. - Increasing the concentration of substrate does not completely overcome inhibition. - Examples: Heavy metal ions Pb2+, Ag+, and Hg2+. Aki | 2  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Irreversible Inhibition  Addition of the phosphate (phosphorylation) catalyzed by - An irreversible enzyme inhibitor inactivates enzymes by a Kinase enzyme forming a strong covalent bond with the enzyme’s active site.  Removal of the phosphate group (dephosphorylation) - The structure is not similar to enzyme’s normal substrate catalyzed by a phosphatase enzyme. - The inhibitor bonds strongly and increasing substrate  Phosphate group is added to (or removed from) the R group concentration does not reverse the inhibition process of a serine, tyrosine, or threonine amino acid residue in the - Enzyme is permanently inactivated. enzyme regulated. - E.g., Chemical warfare agents (nerve gases) and organophosphate insecticides Antibiotics that inhibit Enzyme Activity - An antibiotic is a substance that kills bacteria or inhibits their Regulation of Enzyme Activity growth - Cellular processes continually produce large amounts of an - Antibiotics usually inhibit specific enzymes essential to life enzyme and plentiful amounts of products if the processes are processes of bacteria not regulated. - Two families of antibiotics considered in this discussion are - General mechanisms involved in regulation: sulfa drugs and penicillin’s  Proteolytic enzymes and zymogens covalent modification Sulfa Drugs of enzymes - Many derivatives of sulfanilamide collectively called sulfa  Feedback control Regulation of enzyme activity by various drugs exhibit antibiotic activities substances produced within a cell - Sulfanilamide is structurally similar to PABA (p-aminobenzoic  The enzymes regulated are allosteric enzymes acid) Properties of Allosteric Enzymes - Many bacteria need PABA to produce coenzyme, folic acid  All allosteric enzymes have quaternary structure: - Sulfanilamide is a competitive inhibitor of enzymes responsible - Composed of two or more protein chains for converting PABA to folic acid in bacteria  Have at least two of binding sites: - Folic acid deficiency retards bacterial growth and that - Substrate and regulator binding site eventually kills them  Active and regulatory binding sites are distinct from each - Sulfa drugs don’t affect humans because we absorb folic acid other: from our diet - Located independent of each other Penicillin’s - Shapes of the sites (electronic geometry) are different - Accidently discovered by Alexander Fleming in 1928  Binding of molecules at the regulatory site causes changes in - Several naturally occurring penicillin and numerous synthetic the overall three-dimensional structure of the enzyme: derivatives have been produced - Change in three-dimensional structure of the enzyme leads - All have structures containing a four-membered Beta-lactam to change in enzyme activity ring fused with a five-membered thiazolidine ring - Some regulators increase enzyme activity – activators - Selectively inhibits transpeptidase by covalent modification of - Some regulators decrease enzyme activity – inhibitors serine residue Feedback Control - Transpeptidase catalyzes the formation of peptide cross links - Feedback Control: A process in which activation or inhibition between polysaccharides strands in bacterial cell walls of the first reaction in a reaction sequence is controlled by a Cipro product of the reaction sequence. - The antibiotic ciprofloxacin hydrochloride (Cipro for short) - Regulators of a particular allosteric enzyme may be: - Considered the best broad-spectrum antibiotics because it is  Products of entirely different pathways of reaction within effective against skin and bone infections as well as against the cell infections involving the urinary, gastrointestinal, and  compounds produced outside the cell (hormones) respiratory systems Proteolytic Enzymes and Zymogens - It is the drug of choice for treatment of traveler’s diarrhea - 2nd mechanism of regulating enzyme activity: - Bacteria are slow to acquire resistance to Cipro  Production of enzymes in an inactive form (zymogens) - Biochemical threats associated with terrorism has thrust Cipro into the spotlight because it is effective against anthrax.  Zymogens are “turned on” at the appropriate time and place  Example: proteolytic enzymes: Most digestive and blood- Medical Uses of Enzymes clotting enzymes are proteolytic enzymes - Diagnose certain diseases: Enzymes produced in certain  Hydrolyze peptide bonds in proteins organ/tissues if found in blood may indicate certain damage to  Proteolytic enzymes are generated in an inactive form and that organ/tissue then converted to their active form Covalent Modification of Enzymes General Characteristics of Vitamins - 3rd Mechanism for regulation of enzyme activity  Organic compounds - Covalent modification: A process in which enzyme activity is  Must be obtained from dietary sources altered by covalently modifying the structure of the enzyme:  Human body can’t synthesize in enough amounts  Involves adding or removing a group from an enzyme  Essential for proper functioning of the body - Most common covalent modification: addition and removal of  Needed in micro and milligram quantities phosphate group:  Phosphate group is often derived from an ATP molecule.  1 Gram of vitamin B is sufficient for 500,000 people  Enough vitamin can be obtained from balanced diet Aki | 3  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM  Supplemental vitamins may be needed after illness Vitamin D  Many enzymes contain vitamins as part of their structures - - Two forms active in the body: Vitamin D2 and D3 conjugated enzymes - Sunshine Vitamin: Synthesized by UV light from sun  Two Classes - It controls correct ratio of Ca and P for bone mineralization - Water Soluble and Fat Soluble (hardening)  Synthetic and natural vitamins are same - As a hormone it promotes Ca and P absorption in intestine - 13 Known vitamins Vitamin E - Four forms of Vitamin Es: a-, b-, g- and d-Vitamin E Water- Soluble Vitamins - Alpha-tocopherol is the most active biological active form Vitamin C - Peanut oils, green and leafy vegetables and whole grain - Humans, monkeys, apes and guinea pigs need dietary vitamins products are the sources of vitamin E - Co-substrate in the formation of structural protein collagen - Primary function: Antioxidant – protects against oxidation of - Involved in metabolism of certain amino acids other compounds - 100 mg/day saturates all body tissues - Excess vitamin is Vitamin K excreted - Two major forms; K1 and K2  RDA (mg/day): - K1 found in dark green, leafy vegetables  Great Britain: 30 - K2 is synthesized by bacteria that grow in colon  United States and Canada: 60 - Dietary need supply: ~1/2 synthesized by bacteria and 1/2 obtained from diet  Germany: 75 - Active in the formation of proteins involved in regulating blood clotting NUCLEIC ACIDS - The transfer of genetic information to new cells is accomplished through the use of biomolecules.  Ribonucleic acid (RNA) – found mainly in the cytoplasm Vitamin B of living cells - The preferred and alternative names for the B vitamins  Deoxyribonucleic acid (DNA) – found mainly in the - Thiamin (vitamin B1), Riboflavin (vitamin B2), Niacin nucleus of living cells. (nicotinic acid, nicotinamide, vitamin B3), Vitamin B6 - DNA and RNA are polymers consisting of repeating subunits (pyridoxine, pyridoxal, pyridoxamine), Folate (folic acid), called nucleotides, which are made of three components: Vitamin B12 (cobalamin), Pantothenic acid (vitamin B5), 1. Hetero cyclic base Biotin, Exhibit structural diversity 2. Sugar - Major function: B Vitamins are components of coenzymes 3. Phosphate Fat- Soluble Vitamins Heterocyclic bases Vitamins A, D, E, K - A ring that contains elements other than carbon is called a - Involved in plasma membrane processes heterocyclic ring. - More hydrocarbon like with fewer - The bases found in RNA and DNA contain two types of functional groups heterocyclic rings: pyrimidine and purine. - Vitamin A 1. Pyrimidine Bases: uracil (U), thymine (T), cytosine (C)  Has role in vision - only 1/1000 of vitamin A is in 2. Purine Bases: adenine (A), guanine (G) retina - DNA contains A, G, T, C; RNA replaces T with U  3 Forms of vitamin A are active in the body  Derived from b-carotine Functions of Vitamin A - Vision: In the eye- vitamin A combines with opsin protein to form the visual pigment rhodopsin which further converts light energy into nerve impulses that are sent to the brain. - Regulating Cell Differentiation - process in which immature Sugar and Phosphate cells change to specialized cells with function. - In RNA, the sugar component is D-ribose, and in DNA the  Examples: Differentiation of bone marrow cells white sugar is D-deoxyribose. (Note that both sugars are in the b- blood cells and red blood cells. anomeric form.) - Maintenance of the health of epithelial tissues via epithelial tissue differentiation.  Lack of vitamin A causes such surfaces to become drier and harder than normal. - Reproduction and Growth: In men, vitamin A participates in sperm development. In women, normal fetal development - The phosphate group in nucleotides is derived from phosphoric during pregnancy requires vitamin A. acid, H3PO4, and at physiological pH exists in the ionic form: Aki | 4  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Nucleotides Secondary Structure of DNA - formed from the combination of a sugar with a phosphate group - The bases hydrogen bond to each other in a specific way: A at the 5′ position and a heterocyclic base at the 1′ position. hydrogen bonds to T, and G hydrogen bonds to C, forming a set of complementary base pairs: - ( ′is used to indicate the carbon number in the sugar to distinguish them from the atoms in the bases.) - This allows two separate strands of sugarphosphate backbones to run alongside each other, held together by the hydrogen bonds General Nucleotide Structure between the complementary base pairs: Primary Structure of DNA Double Helix - DNA is one of the largest molecules known, containing - ladder like structure folds in on itself to form double helix, with between 1 and 100 million nucleotide units. the bases on the inside and the sugar-phosphate backbone on the - The nucleotides in DNA are linked by phosphate groups that outside. connect the 5′ carbon of one nucleotide to the 3′ carbon of - The two intertwined polynucleotide chains run in opposite the next. – Because these connections occur on two oxygen (antiparallel) directions, with the 5′ end of one chain on the atoms of the phosphate group, they are called phosphodiester same side as the 3′ end of the other. bonds. - The base sequence of a DNA strand is always written from the - The nucleic acid backbone then is a sequence of sugar- phosphate groups, which differ only in the sequence of bases 5′ end to the 3′ end. attached to the sugars along the backbone (the primary structure - The sugar-phosphate backbone runs along the outside of the of DNA): helix, with the bases pointing inwards, where they form hydrogen bonds to each other. - The two strands of DNA are complementary to each other, because of the specific pairing of G to C and A to T. Discovery of DNA Structure - In 1869, DNA was discovered by the Swiss physician Friedrich Miescher in the pus of discarded surgical bandages; he named it “nuclein” because it was located in the nucleus of the cell. Aki | 5  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM - In 1878, Albrecht Kossel isolated the pure nucleic acid, and - As the replication fork moves down the DNA backbone, the later isolated the five nitrogenous bases. leading strand grows smoothly towards the 5′ end. - Many scientists believed that nucleic acids were far too simple - Since the lagging strand was growing away from the first fork, to be the agent that carried genetic information from one new segments grow from the new location of the replication generation to the next, and that the genetic material would turn fork, until they meet the areas where the RNA primers are out to be a protein. located. - In 1943, Oswald Avery, Colin MacLeod, and Maclyn McCarty - This daughter strand is thus synthesized as a series of fragments identified DNA as the carrier of genetic information. that are bound together in Step 3. The gaps or breaks between - The race was on to determine the structure of DNA, and how it segments in this daughter strand are called nicks, and the DNA was able to transmit genetic information. fragments separated by the nicks are called Okazaki fragments - In 1952, Rosalind Franklin obtained an X-ray crystal structure (after Reiji Okazaki). (“Photo 51”) of a sample of DNA which contained structural Step 3: Closing the nicks. features which lead James D. Watson and Francis H. C. Crick - The daughter strand along the leading strand is synthesized to deduce the double helix structure of DNA (Nobel Prize in smoothly, without any nicks. Medicine, 1962). - The Okazaki fragments along the lagging strand are joined by Chromosomes an enzyme called DNA ligase, which removes the RNA primer - A normal human cell contains 46 chromosomes, each of which and replaces it with the correct nucleotides. contains a molecule of DNA coiled tightly around a group of - The result is two DNA double-helix molecules of DNA that are small basic proteins called histones. identical to the original DNA molecule, each of which contains Genes one old strand from the parent DNA and one new daughter - Individual sections of DNA molecules make up the genes, strand (semiconservative replication). which are the fundamental units of heredity that direct the synthesis of proteins.  Viruses contain a few to several hundred genes.  Escherichia coli (E. coli) contains ~1000 genes.  Humans’ cells contain ~25,000 genes. Replication - the process by which an exact copy of DNA is produced.  Two strands of DNA separate, and each one serves as the template for the construction of its own complement, generating new DNA strands that are exact replicas of the original molecule.  The two daughter DNA molecules have exactly the same base sequences of the parent DNA.  Each daughter contains one strand of the parent and one Step 1: The DNA is unwound by helicase and a replication fork new strand that is complementary to the parent strand. This forms. type of replication is called semiconservative replication. Step 2: With the help of the enzyme DNA polymerase, DNA is DNA Replication replicated smoothly along the leading strand which grows towards Step 1: Unwinding of the double helix. the replication fork. DNA segments (Okazaki fragments) are - The enzyme helicase catalyzes the separation and synthesized by DNA polymerase along the lagging strand as the unwinding of the nucleic acid strands at a specific point replication fork moves. called a replication fork. Step 3: The Okazaki fragments are joined by DNA ligase, resulting - The hydrogen bonds between the base pairs are broken, and in two new DNA molecules. the bases are exposed. - An RNA primer attaches to the DNA at the point where Polymerase Chain Reaction replication begins. - An important laboratory technique called the polymerase chain Step 2: Synthesis of DNA segments. reaction (PCR) mimics the natural process of replication. - A small quantity of target DNA, a buffered solution of DNA - DNA replication takes place from the 3′ end towards the polymerase, the cofactor MgCl2, the four nucleotide building 5′ end of the exposed strands (the template). blocks, and primers are added to a test tube. - Because the strands are antiparallel, the synthesis of new - The primers are short polynucleotides that bind to the DNA nucleic acid strands proceeds: strands and serve as starting points for new chain growth.  toward the replication fork on one strand (the - The mixture goes through several three-step replication cycles: leading strand)  Heat (94-96°C) is used for one to several minutes to  away from the replication fork on the other strand unravel DNA into single strands. (the lagging strand).  The tube is cooled to 50-65°C for one to several minutes - Nucleotides complementary to the ones on the exposed strands to allow primers to hydrogen bond to the separated are attached to the growing chain, and are linked together by the strands of target DNA. enzyme DNA polymerase to form a new daughter strand.  The tube is heated to 72°C for one to several minutes while DNA polymerase synthesizes new strands. Aki | 6  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM - Each cycle doubles the amount of DNA; following 30 cycles, a  the anticodon is a three-base sequence which allows tRNA theoretical amplification factor of 1 billion is attained. to bind to mRNA during protein synthesis. (It is - PCR is a standard research technique that: complementary to one of the codons in mRNA.)  detects all manner of mutations associated with genetic  the 3′ end of the molecule binds to an amino acid with an disease. ester bond and transports it to the site of protein synthesis.  is used to detect presence of unwanted DNA (bacterial An enzyme matches the tRNA molecule to the correct or viral infection). amino acid, “activating” it for protein synthesis.  is a fast and simple alternative to lengthy procedures involving sample cultures that can take weeks. The Central Dogma of Molecular Biology  an be used on degraded DNA samples: - states that genetic information contained in the DNA is - forensic analysis, DNA fingerprinting. transferred to RNA molecules and then expressed in the - Recovery of DNA from extinct mammals, Egyptian mummies, structure of synthesized proteins. and ancient insects trapped in amber to be amplified and - Genes are segments of DNA that contain the information analyzed. needed for the synthesis of proteins. - Each protein in the body corresponds to a DNA gene. Ribonucleic Acid – RNA - a long unbranched polymer consisting of nucleotides joined by 3′ to 5′ phosphodiester bonds. - strands consist of from 73 to many thousands of nucleotides. - Whereas DNA is only found in the nucleus, RNA is found throughout cells: in the nucleus, in the cytoplasm, and in the mitochondria. Transcription, Translation, and Information Flow - Differences in RNA and DNA primary structures: – In RNA the - There are two steps in the flow of genetic information: sugar is ribose instead of deoxyribose. – In RNA, the base uracil 1. Transcription — in eukaryotes, the DNA containing the (U) is used instead of thymine (T). stored information is in the nucleus of the cell, and protein synthesis occurs in the cytoplasm. The information stored in the DNA must be carried out of the nucleus by mRNA. 2. Translation — mRNA serves as a template on which amino acids are assembled in the sequence necessary to produce the correct protein. The code carried by mRNA is translated into an amino acid sequence by tRNA. - The communicative relationship between mRNA nucleotides Secondary structure of RNA and amino acids in a protein is called the genetic code. - Most RNA molecules are single-stranded, although many contain regions of double-helical structure where they form Transcription: RNA Synthesis loops. (A::U, G:::C) - Under the influence of the enzyme RNA polymerase, the DNA double helix unwinds at a point near the gene that is being Kinds of RNA transcribed (the initiation sequence). Only one strand of the Messenger RNA (mRNA) DNA is transcribed. - functions as a carrier of genetic information from the DNA in - Ribonucleotides are linked along the DNA strand in a sequence the cell nucleus to the site of protein synthesis in the cytoplasm. determined by the base pairing of the DNA and ribonucleotide - The bases of mRNA are in a complementary sequence to the bases (A::U, G:::C). base sequence of one of the strands of nuclear DNA. - mRNA synthesis occurs in the 3′ to 5′ direction along the - mRNA has a short lifetime (usually less than one hour); it is synthesized as it is needed, then rapidly degraded to the DNA strand (in the 5′ to 3′ direction along the RNA strand) constituent nucleotides. until the termination sequence is reached. Ribosomal RNA (rRNA) - The newly-synthesized mRNA strand moves away from the - the main component of ribosomes that are the site of protein DNA, which rewinds into the double helix. synthesis. - Synthesis of tRNA and rRNA is similar to this - accounts for 80-85% of the total RNA of the cell. - accounts for 65% of a ribosome’s structure (the remaining 35% is protein). Transfer RNA (tRNA) - delivers individual amino acids to the site of protein synthesis. - specific to one type of amino acid; cells contain at least one specific type of tRNA for each of the 20 common amino acids. - the smallest of the nucleic acids, with 73-93 nucleotides per chain. - has regions of hydrogen bonding between complementary base pairs, separated by loops where there is no hydrogen bonding. - Two regions of tRNA have important functions: Aki | 7  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Introns and Exons Characteristics of the Genetic Code - In prokaryotes, each gene is a continuous segment along a DNA - The genetic code applies almost universally: with minor molecule. exceptions, the same amino acid is represented by the same - Transcription of the gene produces mRNA that is translated into codon(s) in all species. a protein almost immediately, because there is no nuclear - Most amino acids are represented by more than one codon (a membrane separating the DNA from the cytoplasm. feature known as degeneracy). - In eukaryotes, the gene segments of DNA that code for proteins  Only methionine and tryptophan are represented by a single (exons) are interrupted by segments that do not carry an amino codon. acidcode (introns).  Leucine, serine, and arginine are represented by six codons. - Both exon and intron segments are transcribed, producing  No codon codes for more than one amino acid. heterogenous nuclear RNA (hnRNA). - Only 61 of the 64 possible triplets represent amino acids. The - A series of enzymes cut out the intron segments and splice the other three are used as signals for chain termination (a “stop” exon segments together to produce mRNA. signal). - The AUG codon (which also codes for methionine) functions as a “start” signal, but only when it occurs as the first codon in a sequence. Genetic Code - Once the 3D structure of DNA was known, it was clear that the sequence of the bases along the backbone in some way directed Translation and Protein Synthesis the order in which amino acids were stacked to make proteins. - Several ribosomes can move along a single strand of mRNA, - In 1961, Marshall Nirenberg and his coworkers began to producing several identical proteins simultaneously. These unravel the connection between the base sequence in DNA and complexes are called polyribosomes or polysomes. the amino acid sequence in proteins. - The growing polypeptide chain emerging from the end of the - The genetic code uses a sequence of three bases (a triplet code) ribosome spontaneously folds into the characteristic 3D shape to specify each amino acid. (A triplet code gives 43=64 possible of that protein. combinations, which is more than enough to specify the 20 Step 1: Initiation of the polypeptide chain. amino acids.) - mRNA and a small ribosomal subunit join; the initiating codon - Each base triplet sequence that represents a code word on Mrna (AUG) is aligned with P (peptidyl) site of the subunit. molecules is called a codon. - tRNA brings in methionine (eukaryotes) or N-formyl methionine (prokaryotes). - The resulting complex binds to the large ribosomal subunit to form a unit called the initiation complex. Step 2: Elongation of the chain. - The next incoming tRNA with an anticodon that is complementary to the mRNA codon bonds at the A (aminoacyl) site on the mRNA. - A peptide bond is formed between the amino acid segments, (catalyzed by peptidyl transferase), which releases the amino acid chain from the P site. - The “empty” tRNA released, and the whole ribosome moves one codon along the mRNA towards the 3’ end (translocation). - Another tRNA attaches to the A site, and the elongation process is repeated. Step 3: Termination of polypeptide synthesis. - Elongation continues until the ribosome complex reaches a stop codon (UAA, UAG, or UGA). - A termination factor protein binds to the stop codon, and separates the protein from the final tRNA. - The ribosome can then synthesize another protein molecule. Aki | 8  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Mutations Plasmids - any changes resulting in an incorrect base sequence on DNA. - The introduction of a new DNA segment (gene) into a bacterial - Even though the base-pairing mechanism provides a nearly cell requires a DNA carrier called a vector, which is often a perfect way of copying DNA, on average one out of every 1010 circular piece of double stranded DNA called a plasmid. bases are copied incorrectly. – This leads to a change in the - Plasmids range from 2000 to several hundred thousand amino acid sequence in a protein, or causes the protein not to be nucleotides, and are found in the cytoplasm of bacterial cells. made at all. - Plasmids function as accessories to chromosomes by carrying - Mutations occur naturally during replication. They can also be genes for the inactivation of antibiotics and the production of induced by environmental factors: toxins. They are also able to replicate independently of  ionizing radiation (X-rays, UV, gamma rays). chromosomal DNA.  mutagens, which are chemical agents. - A plasmid is isolated from a bacterium, and a restriction enzyme - Mutations may be beneficial to an organism by making it more is added, which cleaves it at a specific site: capable of surviving in its environment, ultimately (over millions of years of accumulating changes) leading to the evolution of new species. - Since much of an organism’s DNA does not code for anything, mutations in these regions are neutral. - Other mutations can be harmful, either producing genetic - When the circular DNA is cut, two “sticky ends” are produced, diseases or other debilitating conditions. which have unpaired bases. Recombinant DNA - The “sticky ends” are provided with complementary sections - produced when segments of DNA from one organism are for pairing from a human chromosome to which the same introduced into the genetic material of another organism. restriction enzyme has been used: - “Genetic engineering” of E. coli to include the gene for the production of human insulin enables large quantities of insulin to be made available for the treatment of diabetes. The Formation of Recombinant DNA - The breaks in the strands are joined using DNA ligase, and the plasmid becomes a circular piece of double-stranded, recombinant DNA. - When the bacteria reproduce, they replicate all of the genes, including the new recombinant DNA plasmids. - Because bacteria multiply quickly, there are soon a large number of bacteria containing the modified plasmid, which are capable of manufacturing the desired protein. Restriction Enzymes - Restriction enzymes, found in a wide variety of bacterial cells, catalyze the cleaving of DNA molecules, except for a few specific types. - These enzymes are normally part of a mechanism that protects certain bacteria from invasion by foreign DNA (such as that in viruses). - In these bacteria, some of the bases in their DNA have methyl groups attached. The methylated DNA of these bacteria is left untouched by the restriction enzymes, but foreign DNA that lacks these bases undergoes rapid cleavage, and is rendered nonfunctional. - Restriction enzymes act at sites on DNA called palindromes, where two strands have the same sequence but run in opposite directions: - Restriction enzymes are used to break DNA up into fragments of known size and nucleotide sequence, which can then be spliced together with DNA ligases. Aki | 9  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Production of Insulin - Gene Therapy: Removal and replacement of defective genes with normal healthy functional genes is known as gene therapy e.g. Sickle cell anemia, Severe Combined Immuno-Deficiency (SCID). SCID is due to a defect in the gene for the enzyme adenosine deaminase (ADA) in 25 per cent of the cases. DIGESTION - Most of the foodstuffs ingested are unavailable to the organism, since they cannot be absorbed by the gastrointestinal mucosa until they are broken down into smaller particles. - The process of changing foodstuffs into simple absorbable forms is called digestion. - This process is accomplished with the aid of hydrolases which Viruses catalyze the hydrolysis of proteins to amino acids and glycerol, - Basic virus particle is called a “virion” – intact and infective and nucleic acids to nucleotides. virus particle - In the course of digestion, minerals and vitamins in the food - Components: Nucleic Acid (DNA or RNA), Protein coat stuffs are also released (capsid) made of individual protein subunits called capsomeres. Classification (Based on foodstuff acted upon) Some may have and outer envelope, a membrane, derived from 1. Amylolytic or carbohydrates-splitting enzymes the host cell. The envelope can have specific spikes of protein  salivary amylase or ptyalin from the salivary glands (H and N spikes of Influenza) that aid in attachment and makes them sensitive to chemical actions of disinfectants.  pancreatic amylase from the pancreas Types of viruses  invertases or disaccharides from the goblet cells of the - based on “morphology” – shape; structure small intestine  Helical (like TMV or Ebola) 2. Proteases or protein-splitting enzymes  Polyhedral (adeno and polio)  (secreted as zymogens or inactive enzymes)  Enveloped (flu)  pepsinogen from the gastric glands of the stomach  Complex (bacteriophage)  trypsinogen and chymotrypsinogen from the pancreas  peptidases from the goblet cells of the small intestine 3. Lipases or fat-splitting enzymes  gastric lipase from the stomach  pancreatic lipase from the pancreas 4. Nucleases or nucleic-acid-splitting enzymes  ribonuclease and deoxyribonuclease from the pancreas Carbohydrates - ptyalin acts on starch in the oral cavity changing starch to maltose - pancreatic amylase on starch in the small intestine changing starch to maltose - maltase acts on maltose changing it to glucose - lactase acts on lactose changing it to glucose and galactose - sucrase acts on sucrose changing it to glucose and fructose Proteins Applications of Recombinant DNA - pepsinogen in the stomach activated to pepsin in the presence - Preparation of gene maps. of HCl, pepsin acts on proteins changing it to peptides - In revealing details of various infections, diseases such as - trypsinogen activated to trypsin by enterokinase in the small "inborn errors of metabolism." intestine and chymotrypsinogen activated to chymptrypsin by - Finding out the complete nucleotide sequence of genome of an trypsin; trypsin and chymotrypsin acts on proteins to change organism and identification of genes. these to peptides - Detecting cytogenetic abnormalities e.g., Down's syndrome, - peptidases act on peptides changing these to amino acids multifactorial disorders, atherosclerosis, coronary artery Lipids disease etc. - gastric lipase in the stomach acts on emulsified fats changing - Preventing various genetic disorders e.g., inherited hemoglobin these to fatty acids and glycerol disorders, phenylketonuria, retinoblastoma etc. - bile in the small intestine emulsifies fats; lipase acts on - Understand a molecular event is biological processes like emulsified fats changing these to fatty acids and glycerol growth, differentiation, ageing etc. Nucleic acids - Replacement or correction of deleterious mutation by transfer - nucleases in the small intestine acts on nucleic acid changing of clone gene in a patient. these to nucleotides - Production of genetically modified organisms (GMOs) or transgenic organisms for providing particular product and nutrient. A k i | 10  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Absorption BIOCHEMICAL ENERGY PRODUCTION - Transport Mechanism Across a Membrane Metabolism  Diffusion - Sum total of all chemical reactions in a living organism  Osmosis - Metabolism will provide the source of energy we need for all  Dialysis our activities such as thinking, moving, breathing, walking,  free diffusion talking, etc.  solvent drag - Energy is also need for many of the cellular processes such as  filtration protein synthesis, DNA replication, RNA transcription and - Carrier-mediated transport transport across the membrane, etc. Catabolism  facilitated diffusion - All metabolic reactions in which large biochemical molecules  active transport are broken down to smaller ones - Bulk transport - Usually, energy is released in these reactions  Phagovytosis - Example: Oxidation of glucose  Pinocytosis Anabolism  emeiocytosis - All metabolic reactions in which small biochemical molecules Sugars are joined to form larger ones - glucose and galactose by facilitated diffusion requiring the - Usually require energy presence of high concentration of external Na ions; - Example: The synthesis of proteins - glucose binds to a carrier molecule which also binds Na and Na Metabolic Pathway moves inward along its concentration gradient dragging glucose - Series of consecutive biochemical reactions used to convert a with it starting material into an end product - fructose does not require Na and its transported by a passive - There are two types of metabolic pathways mechanism along its own concentration gradient  Linear Amino acids  Cyclic - same as glucose and galactose in the absence of a Na gradient, - The major pathways for all forms of life are similar: transport of amino acids will be passive in nature Fatty acids - triglycerides are resynthesized utilizing the partial glycerides or acyloglycerols and the liberated free fatty acids are activated as fatty acyl coA - resynthesized fats pass into the lacteals or lymphatic vessels to large thoracic duct and enters the blood where they are found as Metabolism and Cell structure lipoprotein particles called chylomicrons - Knowledge cell structure is essential to the understanding of Vitamins metabolism - vitamin b12 binds with an intrinstic factor secreted by the - Prokaryotic Cell: Single compartment organism gastric mucosa (absence of this factor in failure of vitamin B12  No nucleus -- found only in bacteria absorption  Single circular DNA molecule present near center of the - folic acid converted to folate before they are absorbed cell called nucleoid - vitamin A binds with retinol-bonding protein before it is - Eukaryotic Cell: Multi-compartment cell absorbed  DNA is present in the membrane enclosed nucleus Inorganic ion transport  Cell is compartmentalized into cellular organelles - Na, K, Ca, Mg, transported by means of carrier molecules  ~1000 times larger than bacterial cells - Diffusion of ions more difficult than diffusion of molecules because passage depends not only on concentration gradient but also on electrical gradient Eukaryotic Cell Organelles and Their Function  Nucleus: DNA replication and RNA synthesis  Plasma membrane: Cellular boundary  Cytoplasm: The water-based material of a eukaryotic cell  Mitochondria: Generates most of the energy needed for cell. A k i | 11  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM  Lysome: Contain hydrolytic enzymes needed for cell rebuilding,  6 Subunit structure: Nicotinamide -- ribose -phosphate -- repair and degradation phosphate - ribose – adenine  Ribosome: Sites for protein synthesis - A typical cellular reaction in which NAD+ serves as the Mitochondria oxidizing agent is the oxidation of a secondary alcohol to give - An organelle that is responsible for the generation of most of a ketone. the energy for a cell: - NAD+: coenzyme  Outer membrane: Permeable to small molecules: 50% lipid, - NADH is reduced form 50% protein  Inner membrane: Highly impermeable to most substances: Coenzyme A 20% lipid, 80% protein - A derivative of vitamin B  Inner membrane folded to increase surface area - Three Subunit Structure  Synthesis of ATP occurs  2-Aminoethanethiol - pantothenic acid - phosphorylated ADP Important intermediate compounds in Metabolic Pathways - Six Subunit structure:  Adenosine Phosphates (AMP, ADP, ATP, cAMP)  2-Aminoethanethiol - pantothenic acid -phosphate -  Monophosphate (AMP): one phosphate group phosphate phosphorylated ribose - adenine  Diphosphate (ADP): Two phosphate groups - Active form of coenzyme A is the sulfhydryl group (-SH group) in the ethanethiol subunit of the coenzyme  Triphosphate (ATP): Three phosphate groups - Acetyl-CoA (acetylated)  Cyclic monophosphate (cAMP): Cyclic structure of phosphate  AMP: Structural component of RNA  ADP and ATP: Key components of metabolic pathways - Phosphate groups are connected to AMP by strained bonds which require less than normal energy to hydrolyze them Classification of Metabolic Intermediate Compounds  The net energy produced in these reactions is used for cellular Metabolic intermediate compounds can be classified into three reactions groups based on their functions - In cellular reactions ATP functions as both a source of a phosphate group and a source of energy.  E.g., Conversion of glucose to glucose-6-phosphate Role of Other Nucleotide Triphosphates in Metabolism - Uridine triphosphate (UTP): involved in carbohydrate High-Energy Phosphate Compounds metabolism - Several phosphate containing compounds found in metabolic - Guanosine triphosphate (GTP): involved in protein and pathways are known as high energy compounds carbohydrate metabolism - High energy compounds have greater free energy of hydrolysis - Cytidine triphosphate (CTP): involved in lipid metabolism than a typical compound: Flavin Adenine Dinucleotide (FAD)  They contain at least one reactive bond -- called strained - A coenzyme required in numerous metabolic redox reactions bond - Flavin subunit is the active form – accepts and donates electrons  Energy to break these bonds is less than a normal bond -- - Ribitol is a reduced form of ribose sugar hydrolysis of high energy compounds give more energy Cellular Reaction than normal compounds - A typical cellular reaction in which FAD serves as oxidizing  More negative the free energy of hydrolysis, greater the agent involves conversion of an alkane to an alkene bond strain - FAD is oxidized form  Typically, the free energy release is greater than 6.0 - FADH2 is reduced form kcal/mole (indicative of bond strain) - In enzyme reactions FAD goes back and forth (equilibrium)  Strained bonds are represented by sign ~ (squiggle bond) from oxidized to reduced form. - NAD+: coenzyme - NADH is reduced form - 3 Subunit stucture:  Nicotinamide - ribose – ADP A k i | 12  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Free Energies of Hydrolosis of Common Phosphate-Containing Stage 2: Acetyl Group Formation Metabolic Compounds - The small molecules from Stage 1 are further oxidized. - End product of these oxidations is acetyl CoA - Involves numerous reactions: Reactions occur both in cytosol (glucose metabolism) as well as mitochondria (fatty acid metabolism) of the cells. Stage 3: Critic Acid Cycle - Takes place in inside the mitochondria - First intermediate of the cycle is citric acid – therefore disgniated as Citric acid cycle - In this stage acetyl group is oxidized to produce CO2 and energy - The carbon oxide we exhale comes primarily from this stage - Most energy is trapped in reduced coenzymes NADH and FADH2 - Some energy produced in this stage is lost in the form of heat Stage 4: Electron Transport Chain and Oxidative Phosphorylation - Takes place in mitochondria - NADH and FADH2 are oxidized to release H+ and electrons - H+ are transported to the inter-membrane space in mittochondria - Electrons are transferred to O2 and O2 is reduced to H2O - H+ ions reenter the mitochondrial matrix and drive ATP- synthase reaction to produce ATP - ATP is the primary energy carrier in metabolic pathways Citric acid cycle - A series of biochemical reactions in which the acetyl portion of acetyl CoA is oxidized to carbon dioxide and the reduced coenzymes FADH2 and NADH are produced - Also know as tricarboxylic acid cycle (TCA) or Krebs cycle: - Citric acid is a tricarboxylic acid – TCA cycle - Named after Hans Krebs who elucidated this pathway - Two important types of reactions:  Oxidation of NAD+ and FAD to produce NADH and FADH2  Decarboxylation of citric acid to produce carbon dioxide Biochemical Energy Production  The citric acid cycle also produces 2 ATP by substrate level - Energy needed to run human body is obtained from food phosphorylation from GTP - Multi-step process that involves several different catabolic - Summary of citric acid cycle reactions: pathways - There are four general stages in the biochemical energy production process:  Stage 1: Digestion  Stage 2: Acetyl group formation,  Stage 3: Citric acid cycle  Stage 4: electron transport chain and oxidative phosphorylation, - Each stage also involves numerous reactions Stage 1. Digestion - Begins in mouth (saliva contains starch digesting enzymes), continues in the stomach (gastric juice), completed in small intestine: Results in small molecules that can cross intestinal membrane into the blood - End Products of digestion:  Glucose and monosaccharides from carbohydrates  Amino acids from proteins  Fatty acids and glycerol from fats and oils - The digestion products are absorbed into the blood and transported to body’s cells A k i | 13  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM Reactions of the Citric Acid Cycle - Several intermediate reactions are involved in this electron  Step 1: Formation of Citrate transfer  Step 2: Formation of Isocitrate Complex II: Succinate-coenzyme Q Reductase  Step 3: Oxidation of Isocitrate and Formation of CO2: involves - Smaller than complex I oxidation–reduction as well as decarboxylation - Contains only four subunits including two iron-sulfur protein  Step 4: Oxidation of Alpha-Ketoglutarate and Formation of clusters (FeSP) CO2 - Succinate is converted to fumarate by this complex  Step 5: Thioester bond cleavage in Succinyl CoA and - In the process it generates FADH2 Phosphorylation of GDP - CoQ is the final recipient of the electrons from FADH2  Step 6: Oxidation of Succinate Complex III: Coenzyme Q – Cytochrom c Reductase - Complex III contains 11 different subunits  Step 7: Hydration of Fumarate - Several iron-sulfur proteins and cytochomes are electron  Step 8: Oxidation of L-Malate to Regenerate Oxaloacetate carriers in this comple Regulation of the Citric Acid Cycle - Cytochrome is a heme iron protein in which reversible - The rate at which the citric acid cycle operates is controlled by oxidation of an iron atom occurs ATP and NADH levels - Various cytochromes, e.g., cyt a, cyt b, cyt c, differ from each - When ATP supply is high, ATP inhibits citrate synthase (Step other by: 1 of Citric acid cycle) - When ATP levels are low ADP, ADP activates citrate synthase  Their protein constituents - Similarly, ADP and NADH control isocitrate dehydrogenase:  The manner in which the heme is bonded to the protein  NADH acts as an inhibitor  Attachments to the heme ring Complex IV: Cytochrome c Oxidase  ADP as an activator. - Contains 13 subunits including two cytochromes Electron Transport Chain - The electrons flow from cyt c to cyt a to cyt a3 - In the final stage of electron transfer, the electrons from cyt a3, - The electron transport chain (ETC) facilitates the passage of and hydrogen ion (H+) combine with oxygen (O2) to form electrons trapped in FADH2 and NADH during citric cycle - ETC is a series of biochemical reactions in which intermediate water carriers (protein and non-protein) aid the transfer of electrons and hydrogen ions from NADH and FADH2 - It is estimated that 95 % of the oxygen used by cells serves as - The ultimately receiver of electrons is molecular oxygen the final electron acceptor for the ETC - The electron transport (respiratory chain) gets its name from the fact electrons are transported to oxygen absorbed via respiration Oxidative phosphorylation - The overall ETC reaction: - process by which ATP is synthesized from ADP and Pi using the energy released in the electron transport chain. - coupled - Energy is used to synthesize ATP in oxidative phosphorylation reactions - Note that 2 hydrogen ions, 2 electrons, and one half-oxygen - Coupled Reactions – are pairs of biochemical reactions that molecule react to form the product water occur concurrently in which energy released by one reaction is - This relatively straight forward reaction actually requires eight used in the other reaction or more steps  Example: oxidative phophorylation and the oxidation - The reaction releases energy (exothermic reaction) reactions of the electron transport chain are coupled - The energy released is coupled with the formation of three ATP systems molecules per every molecule of NADH processed through - The coupling of ATP synthesis with the reactions of the ETC is ETC related to the movement of protons (H+ ions) across the inner - The enzymes and electron carriers needed for the ETC are mitochondrial membrane located along inner mitochodrial membrane - Complexes I, III and IV of ETC chain have a second function - They are organized into four distinct protein complexes and two in which they serve as “proton pumps” transferring protons mobile carriers from the matrix side of the inner mitochondrial membrane to - The four protein complexes tightly bound to membrane: the intermembrane space  Complex 1: NADH-coenzyme Q reductase - For every two electrons passed through ETC, four protons cross the inner mitochondrial membrane through complex I, four  Complex II: Succinate-coenzyme Q reductase through complex III and two more though complex IV  Complex III: Coenzyme Q - cytochrome C reductase - This proton flow causes a buildup of H+ in the intermembrane  Complex IV: Cytochrome C oxidase space - Two mobile electron carriers are: - The gradient build-up would push the H+ ions through  Coenzyme Q and cytochrome c. membrane-bound ATP synthase: Complex 1: NADH-Coenzyme Q Reductase  This high concentration of protons passing through ATP - NADH from citric acid cycle is the source of electrons for this synthase becomes the basis for the ATP synthesis complex - It contains >40 subunits including flavin mononucleotide (FMN) and several iron-sulfur protein clusters (FeSP) - Net result: Facilitates transfer of electrons from NADH to coenzyme Q A k i | 14  BIOCHEMISTRY – LECTURE: 1ST YEAR 2ND SEMESTER MIDTERM A Second Function for Protein Complexes I, III, and IV - > 95% of the ROS formed are quickly converted to nontoxic species in the following reactions: - About 5% of ROS escape destruction by superoxide dismutase and catalase enzymes. - Antioxidant molecules present in the body help trap ROS species ATP Production for the Common Metabolic Pathway - Antioxidants present in the body: - Formation of ATP accompanies the flow of protons from the  Vitamin K intermembrane space back into the mitochondrial matrix.  Vitamin C - The proton flow results from an electrochemical gradient across  Glutathione (GSH) the inner mitochondrial membrane  Beta-carotine - For each mole of NADH oxidized in the ETC, 2.5 moles of ATP - Plant products such as flavonoids are also good antioxidants – are formed. Have shown promise in the management of many disorders - For each mole of FADH2 Oxidized in the ETC, only 1.5 moles associated with ROS production of ATP are formed. - For each mole of GTP hydrolyzed one mole of ATP are formed. - Ten molecules of ATP are produced for each acetyl CoA catabolized Lahat po yan galing sa ppt ni Mam Alfafara (except Nucleic Acid kay sir Jess) copy paste lng lahat yan! Hahahah Review well!! Importance of ATP Goodluckkk – Aki - The cycling of ATP and ADP in metabolic processes is the principal medium for energy exchange in biochemical processes Non-ETC Oxygen Consuming Reactions - >90% of inhaled oxygen via respiration is consumed during oxidative phosphorylation. - Remaining O2 are converted to several highly reactive oxygen species (ROS) with in the body. - Examples of ROS:  Hydrogen peroxide (H2O2)  Superoxide ion (O2-) and  Hydroxyl radical (OH)  Superoxide ion and hydroxyl radicals have unpaired electron and are extremely reactive - ROS can also be formed due to external influences such as polluted air, cigarette smoke, and radiation exposure - Reactive oxygen species (ROS) are both beneficial as well a problematic within the body - Beneficial Example: White blood cells produce a significant amount of superoxide free radicals via the following reaction to destroy the invading bacteria and viruses A k i | 15