Ch 3 Proteins I: Composition and Structures PDF

Ch:3 Proteins I: Composition and Structure Tuesday, November 22, 2022 12:49 PM Functions: provide the matrix (collagen and elastin) for bone and connective tissue. Enzymes. Hemoglobin and myoglobin transport oxygen in blood and store it in muscle, respectively. Transmit compounds and signals acro...

Ch:3 Proteins I: Composition and Structure Tuesday, November 22, 2022 12:49 PM Functions: provide the matrix (collagen and elastin) for bone and connective tissue. Enzymes. Hemoglobin and myoglobin transport oxygen in blood and store it in muscle, respectively. Transmit compounds and signals across membranes. Myosin and actin. Immunoglobulins. Coa gulation proteins. Insulin, thyrotropin, somatotropin (growth hormone), prolactin, luteinizing hormone, and follicle-stimulating hormone are proteins. Regulate gene transcription and translation. Protein is used for molecules that contain over 50 amino acids and peptide is used for those which contain less than 50 amino acids. Important peptide hormones include adrenocorticotropic hormone, antidiuretic hormone, glucagon, and calcitonin. Amino acids: 21 amino acids from 64 codons. Common and derived amino acids. Common amino acids- amino acids for which at least one codon exists in the genetic code. Consists of an alpha-carbon atom, carboxylic acid group, an amino group, hydrogen and a side chain covalently bonded. Derived amino acids- formed by enzymatic modification of the common amino acids. Ex- cystine; desmosine and isodesmosine in elastin; hydroxyproline and hydroxylysine in collagen; carboxyglutamate in prothrombin; and phosphoserine, phosphothreonine, and phosp hotyrosine. Amino acids that contain alkyl group side chains include glycine, alanine, valine, leucine, and isoleucine. Glycine has the simplest structure, with R = H. Phenylalanine, tyrosine, and tryptophan are aromatic amino acids. Tryptophan has indole. Sulfur-containing amino acids are cysteine and methionine. The cysteine side-chain group is a thiolmethyl (HSCH2-). Methionine side chain is a methyl ethyl thiol ether (CH3SCH2CH2- ). The two hydroxy containing amino acids are serine and threonine. Proline is unique and called an alpha-imino acid, since its alpha-amine is a secondary amine in its side chain. Glutamine and asparagine are structural analogs of glutamic acid and aspartic acid with their carboxylic acid side -chain groups amidated. Unprotonated and uncharged. The dicarboxylic-monoamino acids contain a carboxylic group in their side chain. Ex- aspartate and glutamate. At physiological pH, these groups are unprotonated and negatively charged. Class Page 1 unprotonated and negatively charged. The diamino-monocarboxylic acids are lysine, arginine, histidine. Lysine and arginine are protonated at physiological pH and positively charged. In histidine the side chain is the imidazole group. The pKa of the imidazole group is 6.0 in water; thus physiological soluti ons contain high concentrations of both basic (imidazole) and acidic (imidazolium) forms of the group. Selenocysteine: structurally similar to cysteine, but with a selenium replacing sulfur. Identical to one of the termination codons. Thus this codon, UGA, 2 uses. It stops translation signal and also code for selenocysteine incorporation into a protein. Only 25 genes have be en identified that code for proteins that incorporate selenocysteine. The codon is not universal. Many of the selenoproteins have an antioxidant role in eliminating reactive oxygen species. Cystine: derived amino acid. Formed by the oxidation of 2 cysteine thiol side chains to form a covalent disulfide bond. Within proteins, disulfide links of cystine formed from cysteine residues, separated from each other within a polypeptide chain (intrachain) or between two polypeptide chains (interchain), have an important role in stabilizing the folded conformation of proteins like insulin. Optical isomerism: a carbon atom with 4 different substituents in a tetrahedral configuration is asymmetric and exists in 2 enantiomeric forms. All amino acids exhibit optical isomerism except glycine. Fischer projection shows the position in space of the substituents. If the alpha-NH3+ is on the left, the amino acid has an L-configuration (levo) and if the alpha-NH3+ is on the right, it has a D-configuration (dextro). Mammalian proteins contain amino acids of only L-configuration. The L and D refer to the ability to rotate polarized light from its plane of polarization. Formation of peptide bond: dehydration reaction. The alpha-carboxyl group of one amino acid forms a covalent peptide bond with the alpha-amino group of another amino acid by elimination of a molecule of water. Repetition of this process generates the primary structure of the protein. A peptide bond can be represented as 2 resonance structures and the actual peptide bonds are a resonance hybrid of these two isomers, where the carbonyl C-N bond length is approximately half way between that of a C-N single bond and a C=N double bond. A consequence of the partial double bond character is that rotation does not occur about the carbonyl C to N of a peptide bond at physiological temperatures. Second consequence is that all atoms attached to C and N lie in a common plane. Each amino acid residue contributes one alpha-carbon, two single bonds, and a peptide bond to the polypeptide chain. Trans configuration- 2 side chain groups are on opposite side of C=N bond. Most stable. Trans peptide bonds occur in proteins except where there Class Page 2 Trans configuration- 2 side chain groups are on opposite side of C=N bond. Most stable. Trans peptide bonds occur in proteins except where there are proline residues. In proline the side chain includes the alpha-imino group, and both the cis and trans peptide bond configurations with the proline alpha-imino group have unfavorable interactions with the alpha-carbon of the adjacent amino acid forming a peptide bond with the alpha-imino. Cis configuration- 2 side chain groups are on the same side of C=N bond. Unfavorable because of steric hindrance between the side chains. The defined direction of the polypeptide chain is from NH2-terminal amino acid to COOH-terminal amino acid. pH: acid dissociation constant Ka. pKa= log(1/ Ka). pKa depends on the environment. The amino acids whose R groups contain nitrogen atoms (Lys and Arg) are the basic amino acids, since their side chains have high pKa values and are positively charged. Amino acids whose side chains contain a carboxylic acid group have relatively low pKa values and are acidic amino acids. Negatively charged. Proteins in which the ratio (Lys+Arg)/(Glu+Asp) is greater than 1 are basic proteins and those with ratio less than 1 are acidic proteins. Henderson-Hasselbalch equation, pH=pKa+log [conjugate base]/[conjugate acid], shows the change in ionization state and charge of a molecule with pH. Titration of leucine- at pH 1.0 the ionic form (form I) has a charge of +1 and migrates toward the cathode. Addition of 0.5 equivalent of base titrates half the alpha-COOH group; i.e., [COO-]/[COOH]=1 and pH=pKa. Addition of 1 equivalent of base at pH 6.0 completely titrates the alpha-COOH, but it has no effect on the alpha-NH3+ group. In the resulting form (II) , the net charge is zero and is the zwitterion form. Further addition of 0.5 equivalent of base will half-titrate the alpha-NH3+ group. At this point, the ratio of [NH2]/[NH3+]=1, and the pH=pKa. Addition of a further 0.5 equivalent of base (total of 2 full equivalents of base) completely titrates the alpha-NH3+ group to alpha-NH2. The pH becomes greater than 11 and the form has a negative charge. The pH at which an amino acid is in its zwitterion form is the isoelectric pH (pI) for the molecule. The pI value is a consta nt for a compound. For simple amino acid molecules, such as leucine, pI is calculated as the average of the 2 pKa values that regulate the boundarie s of the zwitterion form. pH > pI, then protein charge is negative. pH < pI, then protein charge is positive. The degree of positive or negative charge depends on the magnitude of the difference between pH and pl. Class Page 3 The separation of the plasma proteins is carried out at pH 8.6. The negatively charged proteins move toward the anode. The major fractions are albumin, alpha1-, alpha2-, beta-, and gamma-globulins. Polarity of amino acids: hydrophobicity of amino acid side chains is important for the folding of a protein. The hydrophobic amino acids are buried away from the water solvent. Some nonpolar side chains may be on the surface, where they are generally dispersed among the po lar side chains. When clustering of nonpolar side chains occurs on the surface, it is usually associated with a function, such as to provide a site for binding hydrophobic interactions. Transmembrane proteins reverse the positioning of their side-chain polarity from that of water-soluble globular proteins. Within the membrane, these proteins often position hydrophobic side chains on the outside and ionic groups on the inside to provide binding intera ctions and to form ion channels. An immediate response that occurs with stress or inflammation caused by infection, injury, or surgical trauma, in which haptoglobins in the alpha2 mobility band are selectively increased: A delayed response associated with infection and shows an increase in the gamma-globulin peaks due to an increase in immunoglobulins: Hypo-gamma-globulinemia due to an immunosuppressive disease: In hepatic cirrhosis there is a broad elevation of the gamma-globulins with reduction of albumin: Monoclonal gammopathies are due to the clonal synthesis of a unique immunoglobulin and give rise to a sharp gamma-globulin band: Class Page 4 Nephrotic syndrome shows a selective loss of lower molecular weight proteins from plasma, leading to decrease in albumin but a retention of the bands composed of the alpha2-macroglobulin and beta-lipoproteins in the alpha2 band: Protein-losing enteropathy, who is losing plasma by exudation in the intestinal tract. The slight increase in the alpha2 band is due to an immediate or late response from a stressful stimulus: Chemical reactions: reagents that modify acid side chains have been synthesized to bind to specific sites in a protein's structure, such as the substrate-binding site. The strategy is to model the structural features of the enzyme's natural substrate into the modifying reagent. The reagent binds to the active site like the natural substrate and reacts with a specific side chain. This identifies the modified amino acid as being located in the substrate-binding site and helps identify its role in catalysis. Primary structure: the covalent structure, which includes amino acid sequence and location of disulfide (cystine) bonds. Ex- insulin is initially synthesized as pro insulin, which is a single polypeptide chain with 3 intrachain cystine bonds. Proinsulin is cleaved, which releases two molecules, the C-peptide and insulin, which consists of two polypeptide chains A and B covalently joined by 2 cystine bonds and chain A has an intrachain cystine. Sequence comparisons are used to predict the similarity in structure and function between proteins. Two sequences are homologous when their sequences are highly alignable. Analogy describes sequences from proteins that are structurally similar but for which no evolutionary relationship has been demonstra ted. If a particular amino acid is regularly found at the same position, it is an invariant residue and they have an essential role in structure or function of the protein. Substitution of an amino acid by one of similar polarity (eg, Val for Ile in position 10 of insulin) is a conservative substitution and is observed in sequences of the same protein from different animal species. A nonconservative substitution involves replacement of an amino acid by one of different polarity. This may drastically change the properties of the protein. Other important properties that will significa ntly alter the protein's function are molecular volume and surface area of the residue. Prior to the development of recombinant human insulin, both pig and cow insulins were used in the treatment of human diabetics. Pig insulin is usually more acceptable than cow insulin in insulin-reactive individuals because it is more similar in sequence to human insulin. Proteins have unique secondary, tertiary, and quaternary conformations to produce the protein's native conformation. Folding of the primary structure into the native conformation occurs spontaneously through noncovalent interactions. This conformation is the one of lowest total Gibbs free energy kinetically accessible to the protein. Secondary structure: refers to the folding of the primary structure into helical, pleated sheet, or random conformations. Rotational angles of covalent bonds are located between the nitrogen and alpha-carbon, and the alpha-carbon and carbonyl carbon. The first is the phi bond and the second the psi bond. The third bond is the peptide bond. Due to the partial double-bond character of the carbonyl C-N bonds, there is a barrier to free rotation about this peptide bond. Class Page 5 free rotation about this peptide bond. Regular secondary structure is one where all the phi bond angles are equal, and all psi bond angles are equal. The alpha-helix and beta-strand conformations of polypeptides are the most thermodynamically stable structures. Helical structures are characterized by the no. of residues (n) per turn of helix and the distance (d) between alpha -carbon atoms of adjacent amino acids. The helix pitch (p) is the product of n x d, which measures the distance between repeating turns of the helix. Alpha-helical structure- characteristic are 3.6 amino acid residues per turn (n = 3.6). Each peptide forms 2 hydrogen bonds, one to the peptide bond of the 4th residue above and the other to the peptide bond of the 4th residue below. ln the hydrogen bonds between the peptide groups, the distance between the hydrogen-donor atom and the hydrogen-acceptor atom is 2.9 Å. The side chains are on the outside of the helix. Due to the characteristic 3.6 residues per turn, the 1st and every 3rd and 4th R groups in the helix come close to each other. Helices present polar and nonpolar faces if their amino acid sequences place polar or nonpolar R groups 3 or 4 residues apart. However, if every 3rd or 4th R groups that come together has the same charge or is branched at its beta-carbon, its steric interactions destabilize the helix structure. A right-handed alpha- helix is more stable than a left-handed one. Beta-structure- a polypeptide chain which is hydrogen bonded to another similar polypeptide chain aligned in a parallel or an antiparallel di rection. Hydrogen-bonded beta-strands appear like a pleated sheet. The side chains project above and below this structure. Structural motifs and protein folds- simple arrangements of secondary structure that occur in more than one protein are called structural motifs. They include the helix-turn-helix motif found in many DNA-binding proteins, the strand-turn-strand motif found in proteins with antiparallel beta- structure, and the alternating strand-turn-helix-turn-strand motif found in many alpha/beta-proteins. In these, a turn is a small segment of the polypeptide (3-4 residues) of nonregular secondary structure that connects regions of regular secondary structure. A loop is a bigger segment of connecting nonregular conformation. Combination of motifs forms a fold. A fold is the arrangement of secondary structure elements of a domain. A structural domain is a compact globular structural unit formed within the polypeptide with a hydrophobic core and hydrophilic surface and folds independentl y within the polypeptide chain. Ex- calmodulin domain. Calmodulin binds to target proteins where it acts to sense the calcium level in the cell. At increased calcium levels, calc ium atom binds in calmodulin within the loop of a helix-turn-helix motif called an EF-hand to transmit a signal to its target protein. The fold of the calmodulin domain contains 2 EF-hand motifs interconnected by an alpha-helical segment. Target proteins of calmodulin participate in cell signaling, muscle contraction, fertilization, metabolism, apoptosis, long- and short-term memory, nerve growth, immune response, and cell division. The EF-hand obtained its name from the E and F helices in the muscle protein parvalbumin, in which it was first observed. Tertiary structure: depicts the location of each of its atoms in space. It includes the geometric relationship between distant segments of primar y and secondary structures and the positional relationship of the side chains with one another. Hydrophobic side chains are in the inside, away from the water interface and ionized side chains are on the outside. Within the protein structure are buried water molecules exhibiting stabilizing interactions. A large number of water molecules form a solvation shell around the protein. A long polypeptide folds into multiple domains, each having a hydrophobic core and polar surface. Domains in a multidomain pr otein are connected by a nonregular secondary structure. Ex- trypsin contains 2 domains with a cleft in between that contains the substrate-binding site. Different domains within a protein can move with respect to each other. Ex- hexokinase, which catalyzes phosphorylation of glucose by ATP, has a glucose-binding site in a region between the 2 domains. When glucose binds in the active site, the surrounding domains move to trap i t for phosphorylation. In multifunctional proteins, each domain may perform a different task. Quaternary structure: refers to the arrangement of polypeptide chains in a multichain protein. The subunits in a quaternary structure are associate d noncovalently. Ex- hemoglobin A contains 4 polypeptide subunits (alpha2beta2) held together noncovalently in a specific conformation, and has a quaternary structure. Myoglobin consists of only 1 polypeptide and does not have a quaternary structure. Unstructured proteins: proteins that lack a stable folded structure. Proteins with a nonfolded conformation are called intrinsically unstructured proteins (IUPs). Other proteins may have domains that contain partially unfolded conformations (PUFs). Ex- scaffold proteins, hormones, cyclin- dependent kinases and their inhibitors. The unfolded conformations are highly dynamic. They function by binding to other proteins or to DNA and RNA, which induces a structure in the unfolded polypeptide. This ind uction is a negative entropic process that requires an unfavorable free energy. Thus, the binding strength of unstructured proteins is weak. The l ack of a preformed structure gives them a plasticity to form complementary binding surfaces to many different proteins. Ex- cyclin-dependent kinase inhibitor p21 has the ability to bind to different cyclin-dependent kinases and regulate them. Unstructured regions of proteins can be recognized from their amino acid sequence. Disordered regions are rich in polar and c harged amino acids (glutamate, lysine, and glutamine) and in proline, and lack aromatic and alkyl amino acids. Class Page 6 Complexes, networks and interactomes: protein molecules in cells are present in protein complexes containing multiple protein subunits. The complexes communicate with each other through proteins present in different complexes, which can move between the complexes t o connect them into networks. A complex that interconnects with more than 3 other complexes is a hub in the network, and an important target for drug therapies. A functional network comprising interconnected protein complexes is an interactome. Cellular proteins are characterized using tandem affinity purification (TAP), which involves insertion of a reporter gene in tandem with a gene for a protein of interest to produce a chimeric protein in which the target protein is joined to a tagged protein. The tagged prote in is isolated by elution of the cell lysate over an antitag protein antibody affinity column. The affinity resin binds the tagged protein, which is eluted from the column, separated by electrophoresis, and identified by mass spectrometry. Other methods for identification include co-immunoprecipitation with an antibody directed against the target protein, and the yeast two-hybrid assay in which mammalian protein-protein binary interactions are assessed in a yeast cell reporter system. Bioinformatics: computationally based research area that focuses on the integration and analysis of biodata with computer algorithms. Used to identify patterns within nucleic acid or amino acid sequences that are signatures of motifs and of the protein family or class to which the gene product belongs. Homology searching may be based on structural similarity. X-ray diffraction reveals that the atoms in a protein have fluid-like dynamic motion. It also shows small defects in the packing, indicating the existence of holes for flexibility. Protein domains are classified by class, fold, and family. The class is determined by the predominant type of secondary structure present in the protein. Ex- mainly alpha-helix, mainly beta-strand, and approximately equal amounts of alpha-helix and beta-strand. The fold is determined by the particular arrangement of secondary structure elements within the domain. The family is determined by the degree of sequence identity between the proteins. Proteins that are members of the same family are derived from the same primordial gene. Proteins of the same fa mily have the same fold and similar functions. When the sequence positions involve single changes in the base codon for an amino acid, they are termed single nucleotide polymorphisms (SNPs). Ex- some hemoglobin mutations produce sickle-cell anemia (GAG to GUG). Homologous fold structures: folds of similar structure from unrelated proteins are called superfolds. They form because of the thermodynamic stability of their secondary structure arrangements. Ex- all alpha-fold or domain found in lysozyme called the globin fold. The alpha/beta-domain structure is present in triose phosphate isomerase in which the strands form a central beta-barrel with each beta-strand interconnected by alpha-helical regions located on the outside of the fold. A different type of alpha/beta-superfold is present in the nonhomologous domain 1 of lactate dehydrogenase and domain 2 of phosphoglycerate kinase. An all-beta-domain superfold is present in Cu, Zn superoxide dismutase, in which the antiparallel beta-sheet forms beta-barrel. A similar fold pattern occurs in the domains of immunoglobulins. Concanavalin A shows an all-beta-domain superfold in which the antiparallel beta-strands form a beta- barrel fold called a "jelly roll". Proteins that are not water soluble may contain nonglobular fold patterns. Globular and nonglobular proteins: globular proteins are spherical, vary in size, have high water solubility, and function as catalysts, transporters, and regulators. Fibrous and membrane proteins are nonglobular and have low water solubility. Fibrous proteins contain larger amounts of regul ar secondary structure, a long cylindrical shape, and a structural role. Ex- collagen, keratin, and tropomyosin. Lipoproteins and glycoproteins contain lipid and carbohydrate nonprotein components and may or may not have globular structur es. Collagen: skin collagen is rich in glycine, proline, hydroxyproline, and hydroxylysine. Collagens are glycoproteins with carbohydrate joined to hydroxylysine by an O-glycosidic bond. Each mature collagen molecule contains 3 polypeptide chains. In type 1, there are 2 alpha-1(l) chains and 1 alpha-2(I). Type V collagen contains alpha-1 (V), alpha-2(V), and alpha-3(V) chains. In all types there are regions with the tripeptides Gly-Pro-Y and Gly-X-Hyp repeated in tandem several times. Structure- the poly-Pro forms a helix with 3 residues per turn. This helix is the polyproline type II helix. The three chains of a collagen molecule form a superhelix. It is stabilized by the interchain hydrogen bonds between the 3 chains, because glycine occurs at every third position and forms an apolar edge, which then forms nonpolar interactions between the chains. In type I collagen, only the carboxyl-terminal and amino-terminal segments (known as the telopeptides) are not in a triple-helical conformation. Class Page 7 Covalent cross-links- an enzyme acts on procollagen molecules to convert the amino group of lysine side chains to an aldehyde. The derived amino acid is allysine, which reacts with other allysine groups to form covalent linkages. These linkages can be between chains within the superhel ical structure or between adjacent superhelical collagen molecules in a collagen fibril. Elastin: gives tissues and organs the ability to stretch without tearing. Found in ligaments, lungs, walls of arteries, and skin. It l acks a regular secondary structure. Enzyme lysine amino oxidase converts lysine side chains to allysines. 3 allysines and an unmodified lysine form the heterocyclic structure of desmosine or isodesmosine, which cross-link the polypeptide chains. Keratin and tropomyosin: fibrous proteins in which each polypeptide is alpha-helical. Keratin is found in the epidermal layer of skin, in nails, and in hair. Tropomyosin is a component of actin. Their sequences show tandem repetition of 7 residues (heptad), in which the 1st an d 4th amino acids have hydrophobic side chains and the 5th and 7th have polar side chains. Symbolically represented by (a-b-c-d-e-f-g). The apolar residues form an apolar edge, which interacts with other apolar edges to form a coiled-coil super helix. Each polypeptide also contains a polar edge that interacts with water on the outside and stabilizes the superhelix. Class Page 8 polar edge that interacts with water on the outside and stabilizes the superhelix. Plasma lipoproteins: lipids and proteins are held together noncovalently. It transports lipids from tissue to tissue and participates in lipid met abolism. Their protein components are termed apolipoprotein. The most prominent apolipoproteins are ApoA-1 in high-density lipoproteins (HDLs), ApoB in low-density lipoproteins (LDLs), intermediate-density lipoproteins (IDLs), and very-low-density lipoproteins (VLDLs), and ApoC in IDLs and VLDLs. Structure of VLDL- on the inside are neutral lipids such as cholesteryl esters and triacylglycerols. On the outside is a shell consisting protei ns and charged amphoteric lipids such as unesterified cholesterol and phosphatidylcholines. Smaller particles (HDLs) have a higher percentage of surface proteins and amphoteric lipid molecules than the larger particles (VLDLs and chylomicrons). Structure of apolipoproteins- they have high alpha-helical content when associated with lipids. Their helical regions are amphipathic since every 3rd or 4th amino acid is charged. These residues form a polar edge. The opposite sides of the helices have hydrophobic side chain s that are directed toward their core. Ex- ApoB contains alpha-helical and beta-strand segments. Glycoproteins: amino acids covalently bonded to carbohydrate. Those on plasma membranes help in cell to cell interactions and for regulation of cell growth by contact inhibition. Provide the antigenic determinants of blood groups (e.g., ABO and Rh). The major plasma protein s except albumin are glycoproteins. Covalent linkages- the 2 most common carbohydrate linkages are the N-glycosidic linkage (type I linkage) between the amide group of asparagine and a sugar, and the O-glycosidic linkage (type II linkage) between a hydroxyl group of serine or threonine and a sugar. In type l the bond is to asparagine within the sequence Asn-X-Thr(Ser). O-glycosidic bonds to 5-hydroxylysine is type Ill linkage, to the hydroxyl group of 4-hydroxyproline is type IV linkage, to a cysteine thiol is type V linkage, and to an alpha-amino group of a polypeptide chain is type VI linkage. High concentration of type VI linkages are formed nonenzymatically with hemoglobin and blood glucose in uncontrolled diabetes. Glycosylated hemoglobin: designated HbA1c, is formed nonenzymatically in RBCs by combination of the NH2 terminal amino groups of the hemoglobin beta chains and glucose. The aldehyde form of glucose first forms a Schiff base with the amino group, which then r earranges to a more stable amino ketone linkage, by a reaction known as the Amadori rearrangement. The concentration of HbA1c depends on the concentration of glucose in the blood. Patients with diabetes mellitus have high concentrations of blood glucose and therefore high amounts of HbA1c. Hyperlipoproteinemias: disorders of the synthesis or clearance of lipoproteins from the bloodstream. Detected by measuring plasma triacylglycerol and cholesterol levels. Type I- due to accumulation of chylomicrons. 2 genetic forms are lipoprotein lipase deficiency and ApoCII deficiency. Suffer from eruptive xanthomas (yellowish triacylglycerol deposits in the skin) and pancreatitis. Type ll- elevated LDL levels due to genetic defects in the synthesis of LDL receptor. May suffer myocardial infarctions. Type III- due to abnormalities of ApoE, which interfere with the uptake of chylomicron and VLDL. Increased risk of atherosclerosis. Type IV- commonest abnormality. The VLDL levels are increased due to obesity, alcohol abuse, or diabetes. Type V- associated with high chylomicron triacylglycerol levels, pancreatitis, with eruptive xanthomas. Hypercholesterolemia occurs in certain types of liver disease in which biliary excretion of cholesterol is reduced. An abnormal lipoprotein called lipoprotein X accumulates. Hypolipoproteinemias: abetalipoproteinemia is a genetic disease characterized by absence of chylomicrons, VLDLs, and LDLs due to an inability to synthesize apolipoproteins ApoB-100 and ApoB-48. There is accumulation of lipid droplets in small intestinal cells, malabsorption of fat, acanthocytosis (spiny-shaped red cells), and neurological disease (retinitis pigmentosa, ataxia, and retardation). Tangier disease, an alpha-lipoprotein deficiency, is an autosomal recessive disease in which the HDL level is 1- 5% of its normal value. The accumulation of cholesterol in the lymphoreticular system leads to hepatomegaly and splenomegaly. The plasma cholesterol and phospholipids are greatly reduced. Deficiency of the enzyme lecithin:cholesterol acyl transferase results in the production of lipoprotein X, decrease in the alpha-lipoprotein and pre- beta-Iipoprotein bands and increase in beta-lipoprotein because of the presence of lipoprotein X. Class Page 9 Folding of proteins: polypeptide sequence contains the information for folding. Chaperone proteins facilitate protein folding. The conformation o f a protein is one of lowest Gibbs free energy accessible to its sequence. Folding is under both thermodynamic and kinetic cont rol. Side chains that promote folding are called initiation sites. They form secondary structures and interact with each other to form a molten-globule state. These regions are highly mobile. The rate-determining step for folding and unfolding of the native conformation lies between the molten globule and the native structur e. Chaperone proteins: proteins that facilitate folding include cis-trans-prolyl isomerases, protein disulfide isomerases and chaperone proteins. Cis- trans-prolyl isomerases interconvert cis and trans peptide bonds of proline residues. Protein disulfide isomerases catalyze the bre akage and formation of disulfide cystine linkages. Chaperone proteins were discovered as heat shock proteins (hsps). Chaperones of the 70-kDa family bind to polypeptides as they are synthesized to shield it. Some proteins cannot complete their folding process in the presence of hsp70 chaperones and are sent to the hsp60 family, also called chaperonins. The chaperonins are long cylindrical multisubunit structures that bind unfolded polypeptides in their molten -globule state within their central hydrophobic cavity using ATP. They help in refolding of proteins, and facilitate protein transport into mitochondria and ER. Non covalent forces: cause a polypeptide to fold into its native conformation and then stabilize this structure against denaturation. Noncovalent forces are weak bonding forces. Hydrophobic forces- a nonpolar side chain dissolved in water induces a solvation shell of water. When 2 nonpolar side chains come together, the surface area exposed to solvent is reduced and some of the water molecules in the solvation shell are released to bulk solven t. The entropy of the system is increased and is the driving force causing nonpolar molecules to come together. In transition from random to regular secondary conformation, one-third of the water of solvation is lost to the solvent. Another one-third is lost when a polypeptide attains its native conformation. This brings different segments into close proximity with each other. Hydrogen bonds- formed when a hydrogen atom covalently bonded to an electronegative atom is shared with another electronegative atom. The atom to which the H is bonded is the hydrogen donor atom, and the atom with which the hydrogen atom is shared is the hydrogen acceptor atom. The strength of a hydrogen bond depends on the distance between donor and acceptor atoms. Bonds of higher energy are collinea r. Hydrogen bonds contribute to thermodynamic stability. Electrostatic interactions (ionic or salt linkages)- help in stabilization of proteins and in binding of charged ligands. They can be repulsive or attractive. Its strength directly depends on the charge of each ion and inversely depends on the dielectric constant of the s olvent and the distance between the charges. van der Waals forces- weakest of the noncovalent forces. They have an attractive term inversely dependent on the 6th power of the distance between 2 atoms, and a repulsive term inversely dependent on the 12th power of this distance. The repulsive force is commonly called steric hindrance. The distance of maximum interaction between 2 atoms is the van der Waals contact distance, which is the sum of van der Waals radii for the 2 atoms. The van der Waals repulsive forces between atoms of a peptide bond are weakest at the specific phi and psi angle s compatible with the alpha-helix and beta-strand structures. So, the absence of van der Waals repulsive force is critical for secondary structure formation in proteins. pi-electron-pi-electron interaction occurs when electrons of two aromatic rings interact with each other. Denaturation: occurs when a protein lose its secondary, tertiary, and quaternary structure, but the peptide bonds are not broken. Loss of a hydrogen bond or electrostatic or hydrophobic interaction can lead to destabilization. Protein denaturation occurs on addition of urea, guanidine hydrochloride, detergents, strong base, acid, or heating to above 60°C. Separation based on charge: in electrophoresis, protein is placed in an electric field, where it moves toward the cathode or anode or remains stationary (pH = pI). A technique of extremely high resolution is isoelectric focusing. Ion-exchange column chromatography consists of negatively charged resins which bind to cations called cation-exchange resins, and positively charged resins which bind to anions called anion-exchange resins. Electrophoresis within a silica capillary tube has a high separation efficiency. The wall has a negative charge and an immobile cationic layer. Cations moves toward the cathode in the electric field and causes a flow toward the cathode. Anionic molecules move against this flow , but the current toward the cathode overcomes this, and anions also migrate toward the cathode. Class Page 10 Separation based on size: Ultracentrifugation- a protein subjected to centrifugal force moves in the direction of the force at a velocity dependent on its mass, and its sedimentation coefficient is calculated in Svedberg units. Molecular exclusion chromatography- a porous gel in the form of small insoluble beads is used in column chromatography. Polyacrylamide gel electrophoresis- a charged detergent is added to a protein and electrophoresis occurs through a sieving support. A common detergent is sodium dodecyl sulfate (SDS) and a common sieving support is cross-linked polyacrylamide. The detergent dissociates the quaternary structure of a protein and releases the subunits. Only the molecular mass of the subunits of such proteins are determined by this method. HPLC technique- in high-performance liquid chromatography (HPLC), the solvent passes through a column packed with insoluble polar beadlike resins. It uses high-pressure pumps. Type of chromatography over nonpolar resin beads is called reverse-phase HPLC. Affinity chromatography- proteins have a high affinity for their substrates, receptors, and for antibodies made against them. These compounds can be covalently attached to an insoluble resin, and this resin is used to purify the protein in column chromatography. Purification: activity per unit of protein concentration is called its specific activity. First cell membrane is destroyed, followed by differential centrifugation to isolate the particular protein. Further purification utilizes selective precipitation and then separation b ased on molecular charge, size, and/or affinity. Proteomics- science of determining which proteins are produced in a cell. Mass fingerprinting- mass spectroscopy rapidly determines the amino acid sequence of many small fragments. Determination of amino acid sequence: by Edman reaction or mass spectroscopy. Edman reaction- the polypeptide chain reacts with phenylisothiocyanate. Acidic conditions cleaves the NH2 terminal amino acid as a phenylthiohydantoin derivative, which is separated chromatographically and identified. UV light spectroscopy: side chains of cytosine, phenylalanine, and tryptophan, and peptide bonds absorb UV light and the efficiency of absorption fo r each chromophore is related to its molar extinction coefficient. UV spectroscopy gives information on the secondary and tertiary structures. The molar absorbancy of chromophoric substrate changes on binding to a protein and can be used to measure its binding constant. Fluorescence spectroscopy: in some chromophores, the excitation energy is dissipated by fluorescence. If another molecule is present to absorb light energy emitted by the fluorophore, the emitted fluorescence is transferred to this molecule, and emits its own fluorescence o r loses its excitation energy. If the acceptor molecule loses its excitation energy by nonfluorescent process, it is a quencher of the donor molecules fluorescence. This type of analysis detects changes due to conformational changes and binding interactions. Circular dichroism spectroscopy: caused by differences in light absorption between clockwise and counterclockwise vectors of polarized light passing through an optically active solution such as an L-amino acid, and a spectrum is generated. Class Page 11 Nuclear magnetic resonance: with 2D NMR and powerful NMR spectrometers, the conformation of small proteins can be determined. To determine through-space interactions and tertiary structure nuclear Overhauser effects (NOEs) are used. The major difference between 2D and 1D NMR is the addition of a second time delay rf pulse. A stable α-helix requires hydrogen bonding between peptide bonds at four amino acid intervals. Every peptide bond in the helix participates in this hydrogen bonding. Proline is uncommon in α-helices because it destabilizes the helix by introducing a kink and cannot hydrogen bond with other residues. Other residues with negative (glutamates, aspartates) or positive (lysine, arginine) charges will also destabilize the helix if present in large blocks. Ankyrin mutations that cause spherocytosis disrupt the α-helical domains and interfere with ankyrin stacking that contributes to red cell shape. The altered shape reduces red cell survival, increases hemolysis, and increases the amount of heme converted to bilirubin. Increa sed bilirubin may be seen in the whites of the eyes (sclerae) or skin as a yellow color (jaundice). Increased storage of bilirubin in the gall bla dder may cause gall stones and inflammation (cholecystitis), leading to acute abdominal pain and sometimes requiring gall bladder removal (cholecystecto my). Class Page 12 Ch:9 Proteins II: Structure-Function Relationships in Protein Families Tuesday, November 29, 2022 11:49 PM Immunoglobulins: antibodies are produced by lymphocytes in response to foreign particles. Molecules that induce antibody production are called antigens. A hapten is a small molecule that cannot alone elicit production of specific antibodies but when attached to a larger molecule, it induces antibody synthesis. Antibodies are glycoproteins composed of 2 light chains and 2 heavy chains [(LH)2]. The 4 chains are covalently interconnected by disulfide bonds. Sequences of the NH2-terminal of L and H are the variable (V) regions and they contain subregions called hypervariable regions. They are the complementarity-determining regions (CDRs) as they form the 3D antigen-binding site complementary to the antigen. The COOH-terminal of L and H chains are the constant (C) regions. The CH regions provide for binding of proteins, and contain the site necessary for antibodies to cross the placental membrane. The V regions determine the antigen specificity of the antibody. lgA class are dimers and IgM are pentamers. The different H chains are designated gamma, alpha, delta, mu and epsilon in lgG, IgA, lgM, lgE, and lgD classes, respectively. 2 types of L chains are designated lambda and kappa. IgG is the major immunoglobulin in plasma. Homologous 3D domains- within each chain is a repeating pattern of amino acid sequences and each repeat contains an intrachain disulfide bond. Each amino acid segment has an arrangement of anti-parallel beta-strands called an immunoglobulin fold. 2 immunoglobulin folds form globular domains. Hinge region connects the two VH-CH1 domains with the CH2-CH2 domain of the H chains. So, an antibody has 6 domains. The binding site of the domains is composed of hypervariable sequences. There are 2 NH2-terminal VL-VH domains. The antigen-binding site is demonstrated by treating antibody with the enzyme papain, which hydrolyzes a peptide bond in the hinge region to release 3 fragments, of which 2 are identical and are called antigen binding fragments (Fab) and the other is called the crystallizable fragment (Fc), which does not bind the antigen. Major features of antibody structure and antibody-antigen interactions are: 1. Repeating homologous sequences in L chains form 2 immunoglobulin folds and in the H chains 4 folds. 2. Immunoglobulin folds on separate chains form 6 domains. 3. The antigen-binding site is formed by hypervariable loops (CDRs) in each VL-VH domain. 4. The interactions between antigen and antibody CDRs are non-covalent. 5. Conformational changes occur in the VL-VH domain on binding of antigen, which induces changes in distant domains that alter the binding affinity of effector sites in the constant domains. Immunization: vaccine consists of inactivated pathogen or recombinant proteins. Hemoglobin: globular proteins. They transport oxygen from the lungs to cells. They also transport CO2 and H+ from the cells to the lungs, and release nitric oxide (NO) in the blood vessels of the tissues. NO is a vasodilator and inhibitor of platelet aggregation. A hemoglobin molecule consists of 4 polypeptide chains and each chain contains a heme prosthetic group that binds oxygen to form Hb(O2)4. The major form in adults, HbA1, consists of 2 alpha and 2 beta chains. The fetal form, HbF contains the same alpha chains as HbA1, and gamma chains. Two other hemoglobin forms appear in the first months in which the alpha chains are substituted by zeta sequence and the epsilon chains serve as the beta chains. A minor adult hemoglobin HbA2 contains 2 alpha and 2 delta chains. Class Page 13 Myoglobin: O2 binding protein that releases O2 in skeletal muscle cells. They contain only a single polypeptide chain and 1 O2 binding site. Heme group: a prosthetic group is a nonpolypeptide molecule that forms a functional part of a protein. Without a prosthetic group, a protein is called an apoprotein, and with its prosthetic group it is a holoprotein. In hemoglobins and myoglobin, the heme is protoporphyrin lX with an iron (ferrous, +2) atom in its center. 4 bonds are to the pyrrole nitrogen atoms of the porphyrin and the 5th bond is to a nitrogen of a histidine imidazole (proximal histidine). In oxy-globins, O2 forms the 6th bond, the O2 being between the ferrous atom and a second histidine imidazole (distal histidine), and in deoxy-globins, the 6th position is unoccupied. The heme is positioned within a hydrophobic pocket of each globin subunit. O2 binding to myoglobin: the association of oxygen with myoglobin is characterized by a equilibrium constant, Keq. Units moles/liter. It is dependent on pH, ionic strength, and temperature. The oxygen-dissociation curve for myoglobin has a rectangular hyperbolic shape. This reflects the fact that myoglobin reversibly binds a single molecule of oxygen. Thus, oxygenated (MbO2) and deoxygenated (Mb) myoglobin exist in a simple equilibrium- Mb + O2 -> MbO2 The equilibrium is shifted to the right (curve to the left) or to the left (curve to the right) as oxygen is added to or removed from the system. A plot of log [Y/(1 - Y)] versus log pO2, according to the Hill equation, yields a straight line with a slope equal to 1 for myoglobin. This is the Hill plot, and the slope (nH) is the Hill coefficient. O2 binding to hemoglobin: it shows positive cooperativity. Cooperative binding of O2 by the 4 subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin tetramer. This is referred to as heme-heme interaction. A plot of Y versus pO2 for hemoglobin is sigmoidal, and a plot of the Hill equation gives a slope (nH) equal to 2.8. If nH=1, no cooperativity. If nH C, the value of B is a constant and the reaction is first order. d[P)/dt = -d[A]/dt = k[A] ln[A] = 2.3 log[A] = -kt + constant At t=0, [Ao] is the initial concentration. ln[A] = -kt + [Ao] 2.3 log[A/Ao] = kt A plot of the In [A] versus t is a straight line with a slope of -k. The half-life (t1/2 or t50%) is the time it would take for 50% of the substrate to be converted to product. t1/2=0.69/k Reversible reactions- At equilibrium, the rate of formation of one component is equal to the rate of formation of the other. d[P]/dt = d[A]/dt k1[A] = k2[P] k1/k2=[P]/[A] The ratio k1/k2 is the equilibrium constant, Keq. Class Page 24 Enzyme-catalyzed reactions: like a second-order reaction. The velocity slows down during the reaction because the concentration of substrate is becoming depleted or product is starting to accumulate. It no longer increases with an increase in [S], as the enzyme and substrate form a complex, and the rate of product formation is proportional to the concentration of the complex. d[P]/dt = k3[ES] where v is the rate of product formation, k is a rate constant, and K is a constant which includes the individual rate constants relating to the substrate's interaction with the enzyme. Michaelis-Menten equation: simplest enzyme reaction is, Class Page 25 If the reaction is irreversible (k4 = 0 or [P] is not present as would be found when enzyme and S are first mixed), where k3 is the catalytic activity of the enzyme (kcat). Since kcat (the rate constant) and [Et] (total enzyme concentration) are constants, kcat[Et] = Vmax (Vm). which is the Michaelis-Menten equation. Km is the concentration of substrate when the velocity of the reaction (v) is one-half of the maximum velocity (Vm), i.e., v/Vm = 0.5, when Km = [S]. Free enzyme concentration- where [Et] is the total concentration of enzyme and [Ef] is the concentration of free enzyme. Km- smaller the value of Km, tighter is the interaction between substrate and enzyme, means it takes less substrate to bind half of the enzyme. Ex- utilization of glucose. Glucose can be phosphorylated by two different kinases to form glucose 6-phosphate. Liver contains both hexokinase and glucokinase that catalyze glucose + ATP -> glucose 6-phosphate + ADP. For hexokinase, the Km for glucose is 0.1 millimolar whereas for glucokinase it is 5 mM. When the concentration of blood sugar is low, hexokinase is used, but when blood glucose increases, glucokinase also functions. Turnover number (kcat)- the no. of molecules of substrate converted to product per unit time per molecule of enzyme. The larger the value of kcat for an enzyme, the faster the reaction will be. Unit is 1/time. When [S] is larger than Km, The velocity becomes independent of the concentration of [S] (slope=0). When [S] is smaller than Km, A plot of v versus [S] is linear only when [S] ATP +AMP. Fructose 1,6-bisphosphatase catalyzes an irreversible reaction that opposes the reaction of 6-phosphofrucro-1-kinase. AMP inhibits fructose 1,6- bisphosphatase, which is opposite to the effect that AMP has on 6-phosphofructo-1-kinase. This increases glycolytic flux by increasing the amount of fructose 1,6-bisphosphate available for the splitting stage. The decrease in lactate that occurs due to the Pasteur effect is readily explained by decreased glycolytic flux. Regulation of 6-phosphofructo-1-kinase by intracellular pH: hydrogen ions rather inhibit 6-phosphofructo-1-kinase. Excessive glycolysis lowers blood pH and leads to an emergency medical situation known as lactic acidosis. Plasma membranes contain a symport for lactate and hydrogen ions that allows transfer of lactic acid into the bloodstream. This defense mechanism prevents pH from getting so low that lactic-acid-producing tissues become pickled. Regulation of 6-phosphofructo-1-kinase by citrate: oxidation of both fatty acids and ketone bodies elevates levels of cytosolic citrate, which inhibits 6-phosphofructo-1-kinase. Class Page 70 Hormonal control of 6-Phosphofructo-1-kinase by cAMP and fructose 2,6-bisphosphate: fructose 2,6-bisphosphate is a positive allosteric effector of 6-phosphofructo-1-kinase and is a negative allosteric effector of fructose 1,6-bisphosphatase.The mechanism uses cAMP as the second messenger of hormone action. Glucagon is released from the a cells of pancreas and circulates in blood until it encounters glucagon receptors on the outer surface of liver plasma membrane. Binding of glucagon to its receptor triggers stimulation of adenylate cyclase through the second messenger cyclic AMP (cAMP), which results in a decrease in fructose 2,6-bisphosphate. This makes 6-phosphofructo-1-kinase less effective and fructose 1,6- bisphosphatase more effective, thereby severely restricting flux from fructose 6-pbosphate to fructose 1,6-bisphosphace in glycolysis. Fructose 2,6-bispbosphare is produced from F6P by the enzyme 6-phosphofructo-2-kinase as a side product rather than as an intermediate of glycolysis. This makes 2 phosphofructokinases for us to contend with, one (6-phosphofructo-1-kinase) produces an intermediate (fructose 1,6- bisphosphate) of glycolysis and the other (6-phosphofructo-2-kinase) produces a positive allosteric effector (fructose 2,6-bisphosphate) of the former enzyme. Fructose 2,6-bisphospbatase opposes 6-phosphofructo-2-kinase by converting fructose 2,6 -bisphosphate co F6P by simple hydrolysis. These kinase and phosphatase activities that determine the amount of fructose 2,6-bisphosphate reside in the same protein, a bifunctional enzyme named 6-phosphofrucco-2-kinase/fructose 2,6-bisphosphatase. cAMP regulates fructose 2,6-bisphosphate levels in liver by activating the phosphatase and inactivating the kinase moieties of the bifunctional enzyme. This is achieved by cyclic-AMP-mediated activation of protein kinase A. Inactive protein kinase A consists of 2 regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunits causes conformational changes that release and activate the catalytic subunits. The activated catalytic subunits then phosphorylate specific serine residues present in many enzymes. Bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase Is regulated by phosphorylation: a single phosphorylation of liver 6- phosphofructo-2-kinase/fructose 2,6-bisphosphatase inactivates the kinase enzyme activity but activates the phosphatase enzyme activity. Dephosphorylation has the opposite effects. This provides a very sensitive mechanism for setting the intracellular concentration of fructose 2,6- bisphosphate in liver cells in response to changes in blood levels of glucagon or epinephrine. Increased levels of glucagon or epinephrine, acting through plasma membrane glucagon receptors and beta-adrenergic receptors, respectively, have the common effect of increasing intracellular levels of cAMP. This activates protein kinase A, which phosphorylates a serine residue of 6- phosphofructo-2-kinase/ fructose 2,6-bisphosphatase which inhibits the kinase activity and activates its phosphatase activity. The resulting decrease in fructose 2,6-bisphosphate makes 6-phosphofructo-1-kinase less effective and fructose 1,6-bisphosphatase more effective and thereby inhibits glycolysis at the level of the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. A decrease in glucagon or epinephrine in the blood results in the opposite effect. A phosphoprotein phosphatase removes phosphate from the bifunctional enzyme to activate the kinase and inactivate the phosphatase. Fructose 2,6-bisphosphate accumulates to a higher steady-state concentration and increases the rate of glycolysis. Thus, glucagon and epinephrine are extracellular signals that stop liver from using glucose, whereas fructose 2,6-bisphosphace is an intracellular signal chat promotes glucose utilization by this tissue. Class Page 71 Insulin opposes the actions of glucagon and epinephrine by means of a signaling cascade initiated by activation of the tyrosine kinase activity of its receptor. Insulin also activates cAMP phosphodiesterase (lowers cAMP levels), inhibits protein kinase A, and activates phosphoprotein phosphatase, all of which oppose the effects of glucagon and epinephrine. Insulin therefore aces to stimulate the race of glycolysis. Class Page 72 Heart contains a different isoenzyme of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphate: although glycolysis is inhibited in liver to conserve glucose for use by other tissues, epinephrine stimulates glycolysis in heart to meet the increased need for ATP caused by an epinephrine-signaled increase in workload. As in liver, epinephrine acts on the heart by way of a beta-adrenergic receptor on the plasma membrane, promoting formation of cAMP. This results in activation of protein kinase A that then phosphorylates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Phosphorylation of the bifunctional enzyme in heart increases rather than decreases fructose 2,6-bisphosphate levels. This is because the isoenzyme of the bifunctional enzyme expressed in heart is different from the isoenzyme expressed in liver. Phosphorylation of the heart isoenzyme occurs at a site that activates rather than inhibits the kinase activity. Increased fructose 2,6-bisphosphate concentration then increases 6-phosphofrueto-1- kinase activity and glycolytic flux. Role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphate in cancer: many cancer cells express a special isoform of 6-phosphofructo-2- kinase/fructose 2,6-bisphosphatase in which the activity of the 2-kinase component greatly exceeds the activity of the 2-phosphatase component. This results in a high steady state concentration of fructose 2,6-bisphosphate which maximally stimulates of 6- phosphofructo-1-kinase activity. This is a major component of the mechanism responsible for the high rate of glycolysis characteristic of rapidly growing tumors. Pyruvate kinase: inhibited by ATP. The liver isoenzyme is greatly activated by fructose 1,6- bisphosphate (FBP), and thereby linking regulation of pyruvate kinase to that of 6-phosphofructo-1-kinase. Thus, if conditions favor increased flux through 6-phosphofructo-1-kinase, the level of FBP increases and acts as a feed-forward activator of pyruvate kinase. The liver enzyme is also regulated by covalent modification by protein kinase A, being active in the dephosphorylated state and inactive in the phosphorylated state. Concurrent inhibition of hepatic glycolysis and stimulation of hepatic gluconeogenesis by glucagon can be explained in part by inhibition of pyruvate kinase caused by activation of protein kinase A by cAMP. Pyruvate kinase, like glucokinase, is induced in liver by high carbohydrate intake and high insulin levels. Role of pyruvate kinase in cancer: cancer cells express a special isoform of pyruvate kinase called PKM2, a splicing variant of the muscle form of pyruvate kinase (PKM1). PKM2 is required for rapid growth of tumor cells. Although PKM1 and PKM2 are closely related, PKM1 cannot substitute for PKM2 in cancer cells. Class Page 73 Class Page 74