CHEM 3511 Exam 1

reactions that dont follow M-M model

1. multisubstrate reactions- reactions that involve 2 substrates [each of the substrates interacts w/ enzyme w/ characteristic Km] [to determine each Km the reaction velocity is measured at diff concentrations of one substrate while the other substrate is present at saturating concentration] [Vmax is max reaction velocity when both substrates are present at concentrations that saturate their binding sites on the enzyme] [if substrates can bind in any order than its a random mechanism, if one must bind before the other then its an ordered mechanism, if one substrate binds and one product is released before the other substrate binds then this is a ping pong mechanism] 2. multistep reactions- each step of the process has forward and reverse rate constants so kcat is a function of many individual rate constants [the meaning of kcat (the enzymes turnover number) is the same as for a single step reaction] 3. nonhyperbolic reactions- allosteric enzymes have multiple active sites so dont obey M-M equation [yields sigmoidal curve so M-M doesnt apply but Km and Vmax can be estimated and used to characterize enzyme activity]

chemical catalytic mechanisms

~(1) acid-base catalysis- proton is transferred b/w enzyme and substrate [acid catalysis is where catalyst acts as acid by donating a proton and the catalyst returns to original form at end of reaction] [base catalysis is where the catalyst accepts a proton and lowers the energy of the transition state so accelerates the reaction] ~(2) covalent catalysis- covalent bond forms b/w catalyst and substrate during formation of transition state [in enzymes that use covalent catalysis an electron rich group in enzyme activate site forms covalent adduct w/ substrate] [enzymes that use covalent catalysis undergo a 2 part reaction process so reaction coordinate diagram contains 2 energy barriers w/ reaction intermediate b/w them] [covalent catalysis is often called nucleophilic catalysis b/c the catalyst is a nucleophile (electron rich group in search of electron poor center the electrophile)] [covalent catalysts accelerate rxns by forming a covalent bond between the enzyme (E) and substrate (S)] ~(3) metal ion catalysis- metal ions participate in reactions by mediating oxidation-reduction reactions or by promoting reactivity of other groups in enzymes active site through electrostatic effects [or can interact directly w/ reacting substrate like stabilizing a negative charge during formation of transition state]

recombinant DNA

~(1) fragment of DNA of appropriate size is generated by action of restriction enzyme (2) fragment is incorporated into another DNA molecule (3) recombinant DNA is introduced into cells where it replicates (4) cells containing desired DNA are identified ~cutting and pasting reactions of endonucleases and ligases allow segment of DNA to be excised from chromosome and inserted into carrier DNA molecule like plasmid that has been cut w/ same restriction enzyme ~antibiotic resistance gene allows selection of cells that have plasmid (only cells that contain plasmid can survive in presence of antibiotic) ~example cloning vector has amp^R gene for resistance to antibiotic ampicillin and gene (lacZ) enconding enzyme ß-galactosidase which catalyzes hydrolysis of galactose derivatives [lacZ has several restriction sites which can be used as insertion point for piece of foreign DNA w/ compatible sticky ends and interrupting lacZ prevents synthesis of ß-galactosidase] ~blue white screening- bacteria w/ intact plasmid have ß-galactoside that cleaves galactose to generate a blue dye [colonies of bacteria where foreign DNA has interrupted lacZ are white] [white colony can then be removed and grown in order to harvest recombinant DNA] [bacteria that lack plasmid entirely will also be white but these cells are eliminated by including ampicillin in culture]

structural proteins (4)

~collagen molecules are assembled in ER and after are trimmed by proteases and align side to side to form fibers [fibers are strengthened by cross links] [collagens dont contain Cys so links dont have disulfide bonds] [instead cross links are covalent bonds b/w side chains that have been chemically modified following polypeptide synthesis] ~one kind of cross link requires the enzyme catalyzed oxidation of 2 Lys side chains which then react to form covalent bond [number and type of cross links increase w/ age which is meat from older animals is tougher than from younger animals]

structural proteins (3)

~collagen- connective tissue within and b/w cells/organs/bone [most abundant animal protein] [except in the extreme N and C terminal regions of polypeptides (which are cleaved off once protein exits the cell) every third amino acid is Gly and about 30% of remaining residues are proline and hydroxyproline (Hyp)] [Hyp residues result from hydroxylation of Pro after polypeptide has been synthesized in reaction that requires ascorbate] [in absence of ascorbate collagen contains too few Hyp residues and hydroxylated lysyl residues so resulting collagen fibers are extremely weak] ~each collagen chain consists of repeating triplets most commonly Gly-Pro-Hyp [the most stable conformation for polypeptide sequence containing repeating units of Gly-Pro-Hyp is narrow left handed helix] [each polypeptide has a left handed twist but the triple helix has right handed twist] ~triple helix has chains that are parallel but staggered by one residue so that Gly appears at every position along axis of triple helix [collagen triple helix is stabilized through H bonding where backbone N-H of each Gly bonds to backbone C=O group in another chain] [geometry prevents other groups from forming H bonds w/ each other but they interact w/ water surrounding triple helix]

metabolic pathways (2)

~(1) metabolic pathways are all connected [substrates are products of other pathways and vice versa] (2) pathway activity is regulated [flux is regulated according to substrate availability and cells need for pathways products] (3) not every cell carries out every pathway (4) each cell has unique metabolic repertoire (5) organisms may be metabolically interdependent ~water soluble vitamins- ascorbic acid (C) [ascorbate] [cofactor for hydroxylation of collagen], biotin (B7) [biocytin] [cofactor for carboxylation reactions], cobalamin (B12) [cobalamin coenzymes] [cofactor for alkylation reactions], folic acid [tetrahydrofolate] [cofactor for one-carbon transfer reactions], lipoic acid [lipoamide] [cofactor for acyl transfer reactions], nicotinamide (niacin, B3) [nicotinamide coenzymes NAD⁺ and NADP⁺] [cofactor for oxidation-reduction reactions], pantothenic acid (B5) [coenzyme A] [cofactor for acyl transfer reactions], pyridoxine (B6) [pyridoxal phosphate] [cofactor for amino group transfer reactions], riboflavin (B2) [flavin coenzymes FAD and FMN] [cofactor for oxidation-reduction reactions], thiamine (B1) [thiamine pyrophosphate] [cofactor for aldehyde transfer reactions] ~fat soluble vitamins- vitamin A (retinol) [light absorbing pigment], vitamin D [hormone that promotes Ca²⁺ absorption], vitamin E (tocopherol) [antioxidant], vitamin K (phylloquinone) [cofactor for carboxylation of blood coagulation proteins]

glycolysis (2)

~(4) aldolase- converts the hexose fructose-1,6-biphosphate to 2 three carbon molecules each of which bears a phosphate group [active site of aldolase contains 2 important residues- Lys that forms Schiff base (imine) w/ substrate and Tyr that acts as base catalyst] [rapid consumption of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate pulls the aldolase reaction forward] ~(5) triose phosphate isomerase- there are 2 products from the aldolase reaction but only glyceraldehyde-3-phosphate proceeds through the remainder of the pathway so dihydroxyacetone phosphate is converted to glyceraldehyde-3-phosphate by triose phosphate isomerase ~(6) glyceraldehyde-3-phosphate dehydrogenase- glyceraldehyde-3-phosphate is both oxidized and phosphorylated [oxidation-reduction reaction where aldehyde group is oxidized and cofactor NAD⁺ is reduced to NADH] [active site Cys participates in reaction and enzyme is inhibited by arsenate which competes w/ Pi for binding in active site] ~(7) phosphoglycerate kinase- removal of phosphoryl group from product of step 6 releases large amount of free energy [the free energy released is used to drive formation of ATP since 1,3-biphosphoglycerate donates its phosphoryl group to ADP] ~(8) phosphoglucerate mutase- 3-phosphoglycerate is converted to 2-phosphoglycerate [phospho-His residue of enzyme transfers its phosphoryl group to 3-phosphoglycerate to generate 2,3-biphosphoglycerate which then gives a phosphoryl group back to the enzyme leaving 2-phosphoglycerate and phospho-His]

glycolysis (3)

~(9) enolase- catalyzes dehydration reaction in which water is elminated [active site includes Mg ion that coordinates w/ OH group and makes it a better leaving group] ~(10) pyruvate kinase- converts phosphoenolpyruvate to pyruvate and transfers phosphoryl group to ADP to produce ATP [occurs in 2 steps- (1) ADP attacks phoshoryl group of phosphoenolpyruvate to form ATP and enolpyruvate (2) tautomerization] ~3 of 10 reactions have large negative ∆G so any of these far from equilibrium reactions could serve as flux control points for glycolysis [the other 7 near equilibrium reactions can accommodate flux in either direction] ~to regenerate NAD⁺ the enzyme lactate dehydrogenase reduces pyruvate to lactate [11th step of glycolysis that allows muscles to function anaerobically] [glucose + 2 ADP + 2 Pi→ 2 lactate + 2 ATP] [lactate can only be converted back to pyruvate or exported from the cell] [when muscle is functioning aerobically NADH produced by glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by oxygen and the lactate dehydrogenase reaction isnt needed] ~pyruvate is still slightly reduced molecule so further catabolism of pyruvate begins w/ its decarboxylation to form a two-carbon acetyl group linked to coenzyme A [resulting acetyl CoA is substrate for citric acid cycle] ~pyruvate isnt always used for catabolism and its carbon atoms provide raw material for synthesizing variety of molecules [can be used to make glucose, fatty acids, oxaloacetate (intermediate in synthesis of amino acids and intermediate in citric acid cycle)]

major features of DNA

~2 polynucleotide strands are antiparallel so phosphodiester bonds run in opposite directions ~DNA ladder is twisted in right handed fashion ~diameter of helix is 20 A and it completes turn about every 10 base pairs (10 base pairs is 34 A) ~twisting of DNA ladder into helix creates grooves of unequal width called major and minor grooves ~sugar phosphate backbone define exterior of helix and are exposed to solvent [negatively charged phosphate groups bind Mg²⁺ cations which helps minimize electrostatic repulsion b/w these groups] ~base pairs are located in center of helix perpendicular to helix axis ~base pairs stack on top of each other so core of helix is solid [although planar faces of base pairs arent accessible to solvent their edges are exposed in major and minor grooves and this allows certain DNA binding proteins to recognize specific bases] ~in nature DNA seldom has perfectly regular conformation b/c of small sequence dependent irregularities

structural proteins (1)

~3 types of cytoskeletal proteins that form fibers in cell- microfilaments, microtubules, intermediate filaments [collagen provides structural support extracellularly] ~microfilaments- polymers of actin that support plasma membrane and determine shape ~ATP is in cleft of actin and the ribose hydroxyl groups and phosphate groups form H bonds w/ protein [F actin is polymerized and in fiber form while G actin is globular] [actin polymer is double chain of subunits in which each subunit contacts 4 neighboring subunits] [each subunit has same orientation so fiber has polarity and end w/ ATP site is (-) end and opposite end is (+) end] ~actin polymerization is driven by hydrolysis of ATP [so most actin subunits in microfilament contain bound ADP and only most recently added still contain ATP] [since ADP and ATP subunits have slightly different conformations proteins can distinguish b/w new and older microfilaments] ~polymerization of actin is reversible process so polymer undergoes constant shrinking and growing as subunits add to and dissociate from both ends of microfilament [treadmilling- when net rate of addition to one end matches rate of removal at other end] [supply of actin monomers to support microfilament growth in one area must come at expense of microfilament disassembly elsewhere] ~capping, branching and severing proteins (along w/ proteins whose activity is sensitive to extracellular signals) regulate assembly/disassembly of actin

enzymes

~3 ways to increase rate of chemical reactions including hydrolysis- (1) increasing temp or adding energy in the form of heat [this accelerates all chemical reactions not just the desired one] (2) increasing concentrations of reacting substances [higher concentrations of reactants increase likelihood that they will encounter each other but space in cell is limited] (3) adding catalyst [substance that participates in reaction yet emerges at end in its original form] ~active sites of nearly all known enzymes are located on crevices on enzymes surface, rate enhancements of 10⁸ or 10¹² are typical of enzymes, even fast reactions are subject to enzymatic catalysis, have reaction specificity [most enzymes are highly specific for their reactants (substrates) and products] [functional groups in active site can distinguish substrates from among many others that are similar in size and shape and can mediate single chemical reaction involving those substrates], activity of many enzymes are regulated so that organism can respond to changing conditions or follow genetically determined developmental programs

DNA as genetic material (1)

~Chargaffs observation- amount of A is equal to amount of T and amount of G is equal to amount of C [total amount of A+G is equal to total amount of C+T] [molecule w/ 2 polynucleotide strands in which A and C in one strand pair w/ T and G in the other] ~each nucleotide includes a nitrogen containing base [the bases A and G are purines while C and T are pyrimidines] [RNA contains U instead of T] ~linking atom N9 in purine and N1 in pyrimidine to a 5 carbon sugar produces a nucleoside [in DNA the sugar is 2'-deoxyribose in RNA the sugar is ribose] [the sugar atoms are numbered w/ primes to distinguish them from atoms of attached bases] ~a nucleotide is a nucleoside where one or more phosphate groups are linked usually at C5' of sugar [AMP,ADP,ATP or dAMP,dADP,dATP based on whether ribose or deoxyribose and how many phosphates are attached] ~in nucleic acid the link b/w nucleotides is called phosphodiester bond b/c single phosphate group forms ester bonds to both C5' and C3' [when nucleotide triphosphate is added to polynucleotide chain a diphosphate group is eliminated- once its incorporated into nucleotide chain its called residue]

acid base chemistry (1)

~H₂O↔H⁺+OH⁻ [aqueous solutions dont actually have H⁺ but H₃O⁺ however H⁺ is delocalized so it exists as larger structure] ~proton doesnt remain associated w/ single water molecule but proton jumps through H bonded network of water molecules [this mobility is higher than mobility of ions that physically diffuse among water so this is why acid base reactions are so fast] ~pure water ionizes only slightly so [H⁺] and [OH⁻] are small [since [H₂O] is much higher than [H⁺] and [OH⁻] its considered constant so Kw=[H⁺][OH⁻]] ~at room temp Kw=10⁻¹⁴ so in pure sample of water [H⁺]=10⁻⁷ and [OH⁻]=10⁻⁷ (since product of [H⁺] and [OH⁻] must be 10⁻¹⁴ a [H⁺]>10⁻⁷ is balanced by [OH⁻]<10⁻⁷ and vice versa) ~when [H⁺]=[OH⁻]=10⁻⁷ then its neutral, when [H⁺]>10⁻⁷ its acidic and when [H⁺]<10⁻⁷ its basic ~pH=-log[H⁺] (difference in one pH unit is 10 fold difference in [H⁺])

DNA sequencing

~Sanger sequencing uses dideoxy nucleotides (nucleotides that lack both 2' and 3' hydroxyl groups) (ddNTP) ~DNA (DNA template) is denatured and mixed with dATP/dCTP/dGTP/dTTP and DNA polymerase [b/c polymerase cant begin a new nucleotide strand but only extend preexisting chain primer is needed] [also includes small amount of all 4 ddNTPs each of which is tagged w/ fluorescent dye] ~as polymerase synthesizes new chain it occasionally adds ddNTPs in place of dNTP which halts further extension of chain since it lacks 3' OH and cant form bond to next nucleotide ~reaction products are subjected to electrophoresis [separated on basis of size where smallest molecules move fastest] [separated molecules are excited w/ laser so residue emits its characteristic color and order of appearance of colors corresponds to order of nucleotides in newly synthesized DNA] [this is the sequence of the elongating strand so the template strand will be its complement] ~pyrosequencing- template DNA is immobilized on plastic surface and primer and DNA polymerase are added [when dNTP is added the pyrophosphate is released and triggers luciferase which creates flash of light] [detector records whether light is produced in presence of particular dNTP] ~b/c DNA samples are too large to be sequenced in single pass the DNA must be broken down into overlapping segments that are individually sequence [sequence of entire DNA is then reconstructed]

enzyme classification

~all biochemical reactions involve addition of some substance to another (or its removal) or the rearrangement of that substance ~(1) oxidoreductases [oxidation-reduction reactions] (2) transferases [transfer of functional groups] (3) hydrolases [hydrolysis reactions] (4) lyases [group elimination to form double bonds] (5) isomerases [isomerization reactions] (6) ligases [bond formation coupled with ATP hydrolysis] ~multiple enzymes catalyzing the same reaction are called isozymes [usually share a common evolutionary origin but differ in their catalytic properties] [various isozymes expressed in diff tissues or at diff developmental stages can perform slightly diff metabolic functions]

free energy changes in metabolic reactions (1)

~all reactions in vivo occur w/ net decrease in free energy so ∆G is always less than zero [metabolic reactions are linked so free energy of thermo favorable reaction can be used to pull second unfavorable reaction forward] ~at equilibrium forward and reverse rates are balanced so there is no net change in concentration of any reactant [when system is not at equilibrium the reactants experience a driving force to reach equilibrium values (the force is ∆G° where ∆G°=-RTlnKeq) (R=8.3145 J/Kmol) (for A+B↔C+D then Keq=[C][D]/[A][B])] ~∆G° is for the standard state while ∆G is a function of the actual concentrations of reactants and the temp [∆G=∆G°+RTln[C][D]/[A][B]] [this shows that criterion for spontaneity is actually ∆G so reaction w/ positive ∆G° may still sometimes proceed in vivo depending on concentrations of reactants in cell] [thermo spontaneity doesnt imply rapid reaction so even substance w/ ∆G<<<0 will usually not react until acted upon by enzyme that catalyzes reaction] ~cleavage of either of ATPs phosphoanhydride bonds is reaction w/ very large negative ∆G° (-30kJ/mol) (ATP+H₂O→ADP+Pi) [when reactions w/ ∆G°>0 are combined w/ ATP hydrolysis reaction they are thermo favorable] ~2 reasons why large amount of free energy is released when ATP is hydrolyzed- (1) ATP hydrolysis products are more stable than reactants [in products ADP and Pi separation of the charges relieves unfavorable electrostatic repulsion] (2) compound w/ phosphoanhydride bond experiences less resonance stabilization than its hydrolysis products

monosaccharides (3)

~anomeric carbon of monosaccharide is the carbonyl carbon in straight chain form and its the carbon bonded to both ring oxygen and hydroxy group in cyclic form [anomeric carbon can undergo oxidation so it can reduce substances] [a free monosaccharide is a reducing sugar while monosaccharide where anomeric carbon has already reacted w/ another molecule is nonreducing sugar] [bond that links anomeric carbon to another group is a glycosidic bond and molecule consisting of sugar linked to another molecule is a glycoside] ~other processes replace hydroxyl group w/ amino group to produce amino sugar [oxidation of carbonyl and hydroxyl groups can yield uronic acids (sugars w/ carboxylic acids)] [can reduce OH to H like what converts to ribose to deoxyribose for DNA synthesis] ~monosaccharide derivatives- (1) NH₃⁺ replaces OH group to make amino sugar (2) oxidation and reduction reactions yield sugars w/ carboxylate groups or additional hydroxyl groups

proteins

~at physiological pH the carboxyl group is unprotonated and the amino group is protonated so amino acid bears both negative and positive charge ~19 out of the 20 standard amino acids are asymmetric so therefore have chirality [all amino acids in proteins are L amino acids not D amino acids] ~hydrophobic amino acids have essentially nonpolar side chains that interact very weakly or not at all w/ water [aliphatic hydrocarbon like side chains of Ala, Val, Leu, Ile and Phe fit into this group as well as Met and Trp since the bulk of the side chain is nonpolar even though it includes atoms that can form H bonds] [Pro is also included but is unique b/c side chain is covalently linked to amino group] [hydrophobic amino acids are almost always located in interior of molecule where they dont interact w/ water and since they lack reactive functional groups they dont participate in chemical reactions]

acid base chemistry (2)

~atmospheric CO₂ dissolves in water- CO₂+H₂O↔H₂CO₃↔H⁺+HCO₃⁻ ~addition of H⁺decreases pH and increases ocean acidity [this could dissolve coral reefs and decrease growth of shell building organisms] ~pH of solution can be altered- adding acid to water increases [H⁺] b/c acid donates proton to water (HCl+H₂O→H₃O⁺+Cl⁻) [adding base increases pH by introducing OH⁻ that recombines w/ existing H⁺ (NaOH+H₃O⁺→Na⁺+2H₂O)] ~most acids/bases dont dissociate completely so final concentration is expressed in terms of equilibrium (Ka=[A⁻][H⁺]/[HA]) [increase in Ka means acid is more likely to ionize] ~pKa=-logKa [high Ka means low pKa and high acid strength] [polyprotic acid has more than one acidic H and has pKa value for each dissociation (first proton dissociates w/ low pKa and subsequent protons are less likely to dissociate and so have high pKas)] ~when acid is added to water the final [H⁺] depends on acids tendency to ionize (HA↔A⁻+H⁺) [pH=pKa+log[A⁻]/[HA] and this allows you to calculate pH of acids that dont completely ionize]

restriction enzymes

~bacteria produce DNA cleaving enzymes known as restriction endonucleases (restriction enzymes) that catalyze breakage of phosphodiester bonds at or near specific nucleotide sequences [bacterial cell protects its own DNA from endonucleolytic digestion by methylating it at same site recognized by its restriction endonucleases] ~restriction enzymes typically recognize 4-8 base sequence that is identical (palindromic) when read in same 5' to 3' direction on both strands [EcoRI creates sticky ends and EcoRV creates blunt ends] ~restriction enzymes digest DNA molecules to yield restriction fragments of predictable size

catalytic constant

~catalytic constant (kcat) describes how fast the ES complex proceeds to E+P [kcat=Vmax/[E]T] ~kcat is the rate constant of reaction when enzyme is saturated w/ substrate (when [ES]=[E]T and when v₀=Vmax) ~kcat is also known as enzymes turnover number b/c its the number of catalytic cycles the each active site undergoes per unit time (or number of substrate transformed to product by single enzyme in given period of time) [turnover number is a first order rate constant] ~enzyme can make reaction happen faster but only when overall change in free energy is <0

metabolism and bioenergetics

~cells break down (catabolize) large molecules to release free energy and small molecules [cells use free energy and small molecules to rebuild larger molecules (anabolism)] [set of all catabolic and anabolic activities constitutes the metabolism] ~fatty acids are used to build triacylglycerols which travel in form of lipoprotein to adipose tissue where they are stored as intracellular fat globules [b/c mass of lipid is hydrophobic and doesnt interfere w/ activities in aqueous cytoplasm fat globule can be enormous occupying most of the volume of the adipocyte] ~amino acids, monosaccharides and fatty acids are known as metabolic fuels b/c they can be broken down by processes that make free energy available for cells activities [in general depolymerization reactions are hydrolytic but in case of glycogen phosphate breaks bonds b/w glucose residues not water so the degradation of glycogen is phosphorolysis (catalyzed by glycogen phosphorylase which releases residues from ends of branches in glycogen polymer)]

analyzing protein structure

~chromatography- size exclusion/gel filtration, ion exchange [DEAE are positively charged beads used and CM are negatively charged] [negatively charged proteins bind to DEAE while neutral and positive pass through and then bound proteins can be dislodged by passing a high salt solution through column or decreasing pH] [isoelectric point pI is the pH where protein carries no net charge] [for molecule w/ 2 ionizable groups the pI lies b/w pKa values of those 2 groups (pI=1/2(pKa1+pKa2))], affinity, HPLC

catalytic triad of chromotrypsin

~chromotrypsin uses both acid base catalysis and covalent catalysis to accelerate peptide bond hydrolysis [these activities depend on three active site residues (His 57, Ser 195 and Asp 102)] [the H bonded arrangement of these residues is called the catalytic triad] [substrates scissile bond (bond to be cleaved by hydrolysis) is positioned near Ser when substrate binds to enzyme] [His abstracts proton from Ser (acting as base catalyst) so O can act as covalent catalyst] [Asp promotes catalysis by stabilizing resulting positively charged imidazole group of His] ~(1) peptide substrate enters active site of chromotrypsin so that scissile bond is close to oxygen of Ser (2) removal of Ser hydroxyl proton by His (base catalyst) allows resulting nucleophilic oxygen (covalent catalyst) to attack carbonyl C of substrate (3) tetrahedral intermediate decomposes when His (acid catalyst) donates proton to N of scissile bond (this cleaves the bond) [Asp promotes reaction by stabilizing His through H bonding] (4) departure of C terminal portion of cleaved peptide leaves N terminal portion of substrate linked to enzyme [this relatively stable complex is known as acyl-enzyme intermediate] (5) water enters active site and donates proton to His (base catalyst) leaving hydroxyl group that attacks carbonyl group of remaining substate (6) in second tetrahedral intermediate His (acid catalyst) donates proton to Ser oxygen leading to collapse of intermediate (7) N terminal portion of original substrate (now w/ new C terminus) diffuses away regenerating the enzyme

myoglobin and hemoglobin (4)

~conformational change alters several ionizable groups in the protein- 2 N terminal amino groups of α subunits and 2 His residues near C terminus of β subunits] [these groups become more acidic and release H⁺ when O2 binds to protein] [Hb-H⁺ + O₂ ↔ Hb-O₂ + H⁺] ~increasing pH of solution of hemoglobin favors O2 binding by pushing reaction the the right and decreasing pH favors O2 dissociation by pushing reaction to left [reduction of hemoglobins oxygen binding affinity when pH decreases is known as the Bohr effect] ~tissues release CO2 in respiration and dissolved CO2 enters blood cells where its converted to bicarbonate (HCO₃⁻) by carbonic anhydrase [CO₂+H₂O↔HCO₃⁻+H⁺] [H⁺ released in this reaction induces hemoglobin to unload its O2 in tissues] ~in lungs high concentration of O2 promotes O2 binding to hemoglobin which causes release of protons which then combine w/ bicarbonate to reform CO2 which is breathed out ~red blood cells use BPG which binds in the central cavity of hemoglobin but only in T state [5 negative charges of BPG interact w/ positively charged groups in T state and stabilizes deoxy conformation] [in R state central cavity is too narrow to accommodate BPG] [w/o BPG hemoglobin would bind O2 too tightly to release it to cells] ~fetal hemoglobin has Ser instead of His (this His has positive charge important for binding BPG in adult hemoglobin) [absence of this interaction reduces BPG binding in fetuses so hemoglobin in fetal red blood cells have higher O2 affinity than adult hemoglobin which helps transfer O2 from maternal circulation across placenta to fetus]

myoglobin and hemoglobin (3)

~cooperative binding is important where in lungs torr is 100 and hemoglobin is 95% saturated where as in the tissues torr is 20 to 40 so it is only 55% saturated [O2 released from hemoglobin is readily taken up by myoglobin in muscle cells since myoglobins affinity for O2 is much higher even at lower oxygen pressure] [myoglobin relays O2 from red blood cells to muscle cell mitochondria where it sustains muscle activity] ~the 4 globin subunits undergo conformational change when they bind O2 [in deoxyhemoglobin (w/o bound O2) the heme Fe ion has 5 ligands so porphyrin ring is dome shaped and Fe lies out of plane] [when O2 binds to produce oxyhemoglobin the Fe (now w/ 6 ligands) moves into center of plane and drags F elix so it moves up culminating in the rotation of one αβ unit relative to the other] [2 states are known as tense (T) (deoxyhemoglobin) and relaxed (R) (oxyhemoglobin)] ~deoxyhemoglobin is reluctant to bind first O2 b/c T conformation is unfavorable for O2 binding [once O2 is bound the tetramer switches to R so subsequent molecules bind w/ higher affinity b/c protein is already in R conformation] [oxyhemoglobin tends to retain O2 molecules until pressure drops significantly- then some O2 is released changing to T so this decreases affinity for remaining bound O2 making it easier for hemoglobin to unload bound oxygen] ~allosteric proteins- proteins w/ multiple binding sites [binding of ligand to one site alters ligand binding affinity of other sites]

additional features of enzymes

~despite similarities in catalytic mechanisms chromotrypsin, trypsin and elastase have diff substrate specificity [chromotrypsin cleaves peptide bonds following large hydrophobic residues, trypsin cleaves after basic residues Arg and Lys, elastase cleaves after small hydrophobic residues like Ala/Gly/Val] [varying specificities are explained by chemical character of specificity pocket (cavity on enzyme surface at active site that accommodates the residue on the N terminal side of scissile bond)] ~activity of proteases is limited by action of protease inhibitors and by synthesizing proteases as inactive precursors (zymogens) that are later activated when and where they are needed [zymogens are activated by proteolysis and enteropeptidase activates trypsinogen by catalyzing hydrolysis of its Lys-Ule bond- the now active trypsin cleaves other zymogens including trypsinogen (autoactivation)] [proteolysis of zymogen elicits small conformational changes that open up substrate specificity pocket and oxyanion hole so enzyme becomes maximally active only when it can efficiently bind its substrates and stabilize transition state] ~protease inhibitors pose as protease substrates but arent completely hydrolyzed [inhibitor remains in active site preventing any further catalytic activity]`\

polysaccharides

~each monosaccharide contains several free hydroxyl groups that can participate in condensation reaction which permits different bonding arrangements and allows for branching ~lactose is disaccharide where anomeric carbon (C1) of galactose is linked to C4 of glucose via β glycosidic bond [in sucrose the anomeric carbon of glucose (α) is linked to anomeric carbon of fructose (β) via α glycosidic bond] ~starch and glycogen are polymers of glucose residues linked by α glycosidic bonds at anomeric C1 of one residue to C4 of next residue [α(1→4)] ~cellulose is linear polymer of glucose residues but the residues are linked by β(1→4) rather than α(1→4) glycosidic bonds [where starch can form compact granules inside cell cellulose forms extended fibers that give rigidity and strength

catalytic efficiency

~effectiveness as catalyst depends on how avidly it binds its substrates and how rapidly it converts them to products [kcat/Km is second order rate constant and indicates how reaction velocity varies according to how often enzyme and substrate combine w/ each other] [value of kcat/Km represents enzymes overall ability to convert substrate to product] ~diffusion controlled limit is maximum rate at which 2 freely diffusing molecules can collide w/ each other in aqueous solution [this limit is achieved by some enzymes so they have reached catalytic perfection b/c its overall rate is diffusion controlled- it catalyzes reaction as rapidly as it encounters its substrate]

enzyme kinetics

~enzyme kinetics- quantify enzymes catalytic power and its substrate affinity as well as its response to inhibitors [the progress of any reaction can be expressed as a velocity (v) of either the rate of disappearance of the substrate (S) or rate of appearance of product (P)] [v=-d[S]/dt=d[P]/dt] [when the enzyme concentration is held constant the reaction velocity varies w/ substrate concentration in a nonlinear fashion (hyperbolic shape suggests that enzyme physically combines w/ its substrate to form enzyme-substrate (ES) complex)] [E+S→ES→E+P] ~as small amounts of substrate are added to enzyme preparation enzyme activity appears to increase linearly [however at very high substrate concentrations enzyme activity levels off as it approaches max value (saturated)] ~all simple enzyme catalyzed reactions yield hyperbolic velocity vs substrate curve but exact shape of curve depends on enzyme, concentrations, concentration of enzyme inhibitors, pH, temp, etc [curves can tell you (1) how fast enzyme operates (2) how efficiently does it convert substrates to products (3) how susceptible is it to inhibitors]

unique properties of enzyme catalysts (1)

~enzymes are large b/c catalytic residues must be precisely aligned in active site so certain amount of surrounding structure is required to hold them in place [enzyme can physically distort substrate as it binds in effect pushing it toward higher energy conformation closer to reactions transition state] ~enzyme increases reaction rate not by binding tightly to substrates but by binding tightly to reactions transition state (substrates that have been strained toward structures of products) [transition state stabilization is accomplished through close complementarity in shape and charge b/w active site and transition state] [nonreactive substance that mimics transition state can bind tightly to enzyme and block its catalytic activity] ~rate acceleration is believed to result from increase in both number and strength of bonds that form b/w active site group and the substrate in transition state ~transition state stabilization in chromotrypsin- (1) oxyanion hole [substrate oxygen can form 2 new H bonds w/ backbone NH groups of Ser and Gly so the backbone NH group preceding scissile bond forms another H bond to Gly] [transition state is stabilized by 3 H bonds that occur b/c carbonyl carbon goes from trigonal planar to tetrahedral geometry] (2) low barrier hydrogen bond [H is shared equally b/w original donor and acceptor atoms [in standard H bond the proton belongs to donor atom and there is energy barrier for its full transfer to acceptor atom]] [low barrier H bond is accompanied by 3x bond strength and stabilization] ~reaction rate (velocity) can be described as disappearance of reactant or appearance of product [for first order reaction v=k[A] (units are s^-1)] [for second order reaction v=k[A][B] (units are M^-1 s^-1)

unique properties of enzyme catalysts (2)

~enzymes increase reaction rates by bringing reacting groups into close proximity so to increase freq of collisions that can lead to reaction [when substrate binds to enzyme their translational and rotational motions are frozen so they can be oriented properly for reaction] ~induced fit- when substrate binds enzymes undergo conformational change so they almost fully enclose substrate in cleft/pocket ~electrostatic catalysis- nonaqueous active site of enzyme allows more powerful electrostatic interactions b/w enzyme and substrate than could occur in aqueous solution [normally glucose in solution is surrounded by ordered water molecules but must be desolvated to fit into active site] [by sequestering substrates in active site enzyme can eliminate energy barrier imposed by ordered solvent molecules thereby accelerating reaction]

enzyme inhibition (3)

~enzymes w/ multiple active sites in one subunit are subject to allosteric regulation [ligand binding may alter activity of other active sites or inhibitor/activator binding may decrease/increase catalytic activity of all subunits] ~feedback inhibitor- when its concentration in cell is sufficiently high it shuts down its own synthesis by blocking an earlier step in its biosynthetic pathway ~factors that influence enzyme activity- (1) change in rate of enzymes synthesis or degradation cal alter amount of enzyme available to catalyze reaction (2) change in location can bring enzyme into proximity to its substrate and increase reaction velocity and vice versa (3) ionic signal like change in pH can activate or deactivate enzyme by altering conformation (4) covalent modification can affect enzymes Km or kcat [most often phosphoryl group or fatty acyl group is added to alter catalytic activity]

monosaccharides (1)

~follow molecular formula (CH₂O)n where n≥3 ~sugar where carbonyl group is an aldehyde is known as aldose and where carbonyl group is ketone is known at ketose [in ketoses the carbonyl group occurs at the C2 carbon] ~named by how many carbons they contain- trioses, tetroses, pentoses, hexoses, etc [glucose is an aldohexose and fructose is a ketohexose] ~monosaccharides have a number of stereoisomers b/c they are chiral compounds (carbon atoms bear four different substituents) [every D sugar is a mirror image of an L sugar where L has H to the right of the carbon and D has H to the left of the carbon] [most naturally occurring sugars have D confirmation so L and D prefixes are often left out] ~some sugars have more than one enantiomeric carbon so there are stereoisomers for the configuration at each of these positions [carbs that differ in configuration at one of these carbons are known as epimers] [galactose is an epimer of glucose at C4]

metabolic pathways (1)

~glycolysis- pathway degrades glucose [the 6 carbon sugar is phophorylated and split in half yielding 2 glyceraldehyde-3-phosphate which is then converted into pyruvate] [the decarboxylation of pyruvate (removal of CO2) yields acetyl-CoA where a two-carbon acetyl group is linked to CoA] [glyceraldehyde-3-phosphate, pyruvate and acetyl CoA are key in many metabolic pathways] [if not used to synthesize other compounds two-carbon intermediates can be broken down to CO2 by the citric acid cycle] ~for metabolic reactions oxidation of carbon frequently appears as replacement of C-H bonds w/ C-O bonds [carbon has given up some of its electrons even though the electrons are still participating in covalent bond] [turning CO₂ into CH₂O requires input of free energy so reduced carbons of carb represent form of stored free energy] ~membrane associated enzyme may transfer electrons from substrate to lipid soluble electron carrier like ubiquinone (Q) [Q can take one or two electrons in contrast to NAD⁺ which can only take 2] [one electron reduction of Q (addition of H) produces semiquinone (stable free radical QH.) and a 2 electron reduction yields ubiquinol (QH₂)] ~catabolic pathways generate lots of reduced cofactors and some of these are reoxidized in anabolic reactions while the rest are reoxidized by synthesis of ATP from ADP and Pi [reoxidation of NADH and QH₂ require reduction of O₂ to H₂O so known as oxidative phosphorylation] ~NAD⁺ and Q collect electrons (free energy) from reduced fuel molecules and when electrons are transferred to O₂ this free energy is harvested in the form of ATP

glycoproteins

~glycoproteins are proteins w/ covalently attached carbohydrates [most common post translational modification and vastly increases diversity of proteins] ~oligosaccharides attached to glycoproteins are usually linked to either an Asn side chain (N linked oligosaccharides) or to Ser/Thr side chain (O linked oligosaccharides) [N glycosylation begins while protein is being synthesized by ribosome] [as protein is translocated oligosaccharide is attached to Asn residue] [when protein leaves ER enzymes known as glycosidases remove various monosaccharide residues and glycosyltransferases add new monosaccharides] ~O linked oligosaccharides are built in Golgi through glycosyltransferases [dont undergo processing by glycosidases] [glycoproteins w/ O linked have many more groups and tend to be longer than N linked so these proteins can be 80% carbohydrate]

myoglobin and hemoglobin (2)

~hemoglobin is tetramer w/ 2 α chains and 2 β chains [each of these subunits globin is very similar to myoglobin- similar structures, all have heme group in hydrophobic pocket, His F8 that ligands the iron ion, His E7 that forms H bond to O₂] ~the amino acid sequences are only 18% identical however [certain tertiary structures can accommodate variety of amino acid sequences and many proteins w/ completely unrelated sequences adopt similar structures] ~invariant residues are essential for structure/function and cant be replaced by other residues, some residues are under less selective pressure and can be conservatively substituted by similar amino acid, variable residues are not critical for structure or function and can be easily substituted ~oxygen carrying capacity of blood can be measured by hematocrit (the percentage of blood volume occupied by red blood cells which ranges from 40% in women to 45% in men) ~plot of Y vs pO₂ for hemoglobin is sigmoidal not hyperbolic and hemoglobins overall oxygen affinity is lower than myoglobin (hemoglobin is half saturated at 26 torr while myoglobin is half saturated at 2.8) [curve is sigmoidal b/c binding of first O2 increases affinity for remaining O2 binding sites (cooperative binding behavior)] [hemoglobins 4 heme groups arent independent but communicate in order to work in unified fashion- 4th O2 binds w/ about 100 times greater affinity than the 1st]

Km and Vmax

~hyperbolic curves are prone to misinterpretation b/c its difficult to estimate upper limit of curve (Vmax) [so to more accurately determine Km and Vmax the data is transformed to a line in velocity vs substrate curve know as Lineweaver-Burk plot] [1/v₀=(Km/Vmax)1/[S]+1/Vmax] ~a plot of 1/v₀ vs 1/[S] gives a straight line whose slope is Km/Vmax and intercept on the 1/v₀ axis is 1/Vmax [the intercept on the 1/[S] axis is -1/Km

secondary structure

~in the peptide bond the electrons are delocalized so peptide bond has 2 resonance forms [due to this partial double bond character there is no rotation around C-N bond] [backbone can still rotate about N-Cα and Cα-C bonds although rotation is limited] ~backbone amide groups are H bond donors and carbonyl O are H bond acceptors [under physiological conditions the polypeptide chain folds so that it can satisfy as many of these H bonding requirements as possible but also adopt conformation that minimizes steric strain] ~there are 3.6 residues per turn of the α helix and for every turn that helix rises 5.4 A along its axis [carbonyl O of each residue forms H bond w/ NH group 4 residues ahead] [most helices are about 12 residues long] [in α helix the side chains of the amino acids extend outward from helix] ~in β sheets each residue forms 2 H bonds w/ a neighboring strand [single sheet can have 2 to 12 strands w/ an average of 6 strands and each strand has average length of 6 residues] [amino acid side chains extend from both faces] ~elements of secondary structure (individual α helices or strands in β sheet) are linked together by loops of various sizes [usually loops are irregular secondary structures b/c they dont have defined secondary structure in which successive residues have same backbone conformation] ~usually 31% of residues are in α helices, 28% in β sheets and the rest in irregular loops of different sizes

enzyme inhibition (1)

~irreversible inhibitors- when compounds interact w/ enzymes so tightly their effects are irreversible [any reagent that covalently modifies an amino acid side chain in protein can potentially act as irreversible enzyme inhibitor] ~suicide substrates- enter enzymes active site and begin to react just as normal substrate would but they are unable to undergo complete reaction and become stuck in active site ~reversible inhibitor may affect enzymes Km, kcat or both [most common form of reversible enzyme inhibition is competitive inhibition (inhibitor is substance that directly competes w/ substrate for binding to enzymes active site)] [usually resembles substrate in size and chemical properties so it can bind to enzyme but lack electronic structure that allows it to react] ~b/c inhibitor prevents some of the substrate from reaching active site the Km increases (enzymes affinity for substrate decreases) [high concentrations of substrate can overcome affect of inhibitor b/c when [S]>>>[I] the enzyme is more likely to bind S than I] [presence of competitive inhibitor doesnt affect kcat so as [S] approaches infinity v₀ approaches Vmax] [competitive inhibitor increases Km of enzyme but doesnt affect kcat or Vmax] ~v₀=Vmax[S]/αKm+[S] where α is factor that makes Km bigger [α=1+[I]/Ki] [Ki is the inhibition constant and its the dissociation constant for EI complex (Ki=[E][I]/[EI]) [the lower the value of Ki the tighter the inhibitor binds to enzyme] ~product inhibition- occurs when product of reaction occupies enzymes active site thereby preventing binding of additional substrate molecules

genomic data

~methods for identifying genes- (1) scan DNA sequence for ORF (stretch of nucleotides that can potentially be transcribed or translated and has start codon and stop codon) (2) sequence comparisons w/ known genes [genes w/ similar functions tend to have similar sequences (homologous)] [genes that appear to lack counterparts in other species are known as orphan genes] ~genome maps indicate placement and orientation of genes on a chromosome [arrows pointing in opposite directions represent genes encoded by different strands of double stranded chromosome] [gene mapping has uncovered horizontal gene transfer which is where gene is transferred b/w species rather than from parent to offspring of same species] ~DNA of any 2 humans differs at 3 million sites or once every thousand base pairs [these are SNPs which are instances where the DNA sequence differs among individuals]

structural proteins (2)

~microtubules are 3 times thicker and more rigid b/c its constructed as a hollow tube [α-tubulin and β-tubulin form a dimer and microtubule grows by addition of tubulin dimers] [each tubulin subunit has nucleotide binding site but binds either GTP or GDP] ~assembly of microtubule begins w/ end to end association of tubulin dimers to form short linear protofilament [protofilaments then align side to side in curved sheet which wraps around itself to form hollow tube] [microtubule extends as tubulin dimers add to both ends but is polar and one end grows more rapidly- the (+) end which terminates in β-tubulin grows about twice as fast as the (-) end which terminates in α-tubulin] ~colchicine causes microtubules to depolymerize blocking cell division [when bound it causes conformational change that weakens lateral contacts b/w protofilaments so microtubules eventually shorten and disappear]

myoglobin and hemoglobin (1)

~myoglobin lacks β structures and has 8 α helices which are 7 to 26 residues [hemoglobin is tetrameric protein whose four subunits each resemble myoglobin] ~heme- type of prosthetic group (organic compound that allows protein to carry out some function that polypeptide cant perform alone) in this case binding oxygen [the heme is wedged into hydrophobic pocket b/w helices E and F of myoglobin] [by itself heme isnt effective oxygen carrier b/c central iron is easily oxidized which cant bind O2- oxidation doesnt take place when heme is part of protein] ~Mb + O₂→ MbO₂ [K=[Mb][O₂]/[MbO₂]] [proportion of total myoglobin molecules that have bond O2 is called fractional saturation (Y)] [Y=[MbO₂]/[Mb]+[MbO₂] or Y=[O₂]/K+[O₂] or Y=pO₂/K+pO₂] [amount of O2 bound to myoglobin (Y) is a function of the oxygen concentration (pO₂) and affinity of myoglobin for O2 (K)] ~plot of Y vs pO₂ yields a hyperbola where as O2 concentration increases more and more O2 molecules bind to heme group of myoglobin until at very high O2 concentration all myoglobin have bound O2 (myoglobin is then saturated w/ oxygen) [oxygen concentration where myoglobin is half saturated is equivalent to K (this is called P50- the oxygen pressure at 50% saturation)] [in human myoglobin p50 is 2.8 torr]

chemistry of catalysis

~normally atoms that approach each other repel each other but if there is sufficient free energy they can pass this point and react w/ each other to form products [horizontal axis is progress of reaction (reaction coordinate) and vertical axis is free energy] [energy requiring step is the free energy of activation or activation energy (∆G≠)] [point of highest energy is the transition state] [the height of the activation energy barrier determines the rate of a reaction] [not all reactants that get together to form transition state actually proceed all the way to products some may return to original state] ~free energy change of reaction (∆G) is G products-G reactants [when G reactants > G products the reaction is spontaneous but when G products > G reactants the reverse reaction proceeds spontaneously] [reaction w/ negative ∆G is thermodynamically favorable but ∆G≠ determines how fast the reaction actually occurs] ~catalyst decreases activation barrier (∆G≠) of reaction by interacting w/ reacting molecules so that they are more likely to assume the transition state [enzyme doesnt alter net free energy change of reaction but merely provides pathway from reactants to products that passes through transition state w/ lower free energy than transition state of uncatalyzed reaction] [lowers height of activation energy barrier by lowering energy of transition state] ~in some cases amino acid side chains of enzyme cant provide required catalytic groups so tightly bound cofactor participates in catalysis [cofactors can be metal ions or coenzymes] [coenzymes can either be cosubstrates which enter and exit the active site as substrates do or prosthetic groups which remain in the active site b/w reactions]

DNA as genetic material (2)

~nucleotides consecutively linked by phosphodiester bonds form polymer in which bases project out from backbone of repeating sugar phosphate groups [end of polymer that bears phosphate group attached to C5' is known as 5' end and end that bears free OH group at C3' end is the 3' end] ~DNA contains 2 polynucleotide strands whose bases pair through H bonding [2 H bonds link A and T and 3 H bonds like C and G] ~all base pairs which consist of purine and pyrimidine have same molecular dimensions so the sugar phosphate backbone of the 2 strands of DNA are separated by constant distance regardless of whether the base pair is A-T, G-C, T-A or C-G

free energy changes in metabolic reactions (2)

~other phosphorylated compounds can give phosphoryl group to another molecule and function as energy currency [phosphoenolpyruvate, 1,3-biphosphoglycerate, ATP→AMP+PPi, phosphocreatine, ATP→ADP+Pi, glucose-1-phosphate, PPi→2Pi, glucose-6-phosphate, glycerol-3-phosphate] ~another class of compounds that can release large amount of free energy upon hydrolysis are thioesters [hydrolysis is more exergonic than of ordinary oxygen ester b/c thioesters have less resonance stability than oxygen esters] [acetyl group linked to thioester can be readily transferred to another molecule b/c formation of this new linkage is powered by the favorable free energy change of breaking the thioester bond]

enzyme classification (2)

~oxidoreductases- all enzymes catalysing oxido-reductions [The substrate oxidized is regarded as hydrogen or electron donor] [classification is based on 'donor:acceptor oxidoreductase'] [ recommended name is 'dehydrogenase' and 'oxidase' is used only where O2 is an acceptor] ~transferases- enzymes transferring a group from one compound to another [classification is based on the scheme 'donor:acceptor grouptransferase'] [the recommended names are normally formed as 'acceptor grouptransferase' or 'donor grouptransferase'] [In many cases, the donor is a cofactor (coenzyme), carrying the group to be transferred] ~hydrolases- enzymes catalyse the hydrolysis of various bonds [systematic name always includes 'hydrolase', the recommended name is, in most cases, formed by the name of the substrate with the suffix -ase] ~lyases- enzymes cleaving C-C, C-O, C-N and other bonds by means other than by hydrolysis or oxidation [two substrates are involved in one reaction direction, but only one in the other direction] [When acting on the single substrate, a molecule is eliminated leaving an unsaturated residue] The systematic name is formed according to 'substrate group-lyase'] ~isomerases- Catalyze geometric or structural changes within a molecule [These enzymes catalyze changes within one molecule] ~ligases- enzymes that catalyze the joining of two molecules with concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate ['Ligase' is commonly used for the recommended name but in a few cases 'synthase' or 'carboxylase' is used]

Michaelis-Menten equation

~unimolecular reaction is one that involves single reactant (A→B) [described by rate equation in which the reaction rate (velocity) is expressed in terms of rate constant and reactant concentration [A]] [v=-d[A]/dt=k[A]] [k is the rate constant and has units s⁻¹] [velocity is directly proportional to [A] so it is first order b/c its rate depends on concentration of one substance] ~bimolecular reaction involves 2 reactants and is second order reaction (A+B→C) [v=-d[A]/dt=-d[B]/dt=k[A][B]] [k is second order constant and has units of M⁻¹s⁻¹] [velocity is proportional to product of the 2 reactant concentrations] ~enzyme binds substrate before converting it to product so overall reaction consists of first order and second order processes each w/ characteristic k [E+S↔ES→E+P] [initial E+S is bimolecular w/ k₁ and then ES can either undergo unimolecular to E+P (k₂) or unimolecular back to E+S (k⁻₁)] [rate equation for product formation is v=k₂[ES]] ~Michaelis-Menten equation is the rate equation for enzyme catalyzed reaction and is the mathematical description of the hyperbolic curve [v₀=Vmax[S]/Km+[S]]

glycolysis (1)

~pathway converts 6 carbon glucose into 3 carbon pyruvate in 10 steps [(1) each step of the pathway is catalyzed by a distinct enzyme (2) free energy consumed or released in certain reactions is transferred by molecules like ATP and NADH (3) rate of pathway can be controlled by altering activity of individual enzymes] ~glucose+2 NAD⁺+2 ADP+2 Pi→2 pyruvate+2 NADH+2 ATP ~2 ATP are consumed in the energy investment phase but 4 ATP are produced w/ a net gain of 2 ATP [each glucose molecule that enters the pathway yields 2 three-carbon molecules] ~(1) hexokinase- transfers phosphoryl group from ATP to C6 OH group of glucose to form glucose-6-phosphate [kinase is enzyme that transfers phosphoryl group from ATP to another substance] [hexokinase catalyzes a metabolically irreversible reaction that prevents glucose from backing out of glycolysis (many pathways have irreversible step near starts that commits metabolite to proceed through pathway)] ~(2) phosphoglucose isomerase- isomerization reaction where glucose-6-phosphate is converted to fructose-6-phosphate [value of ∆G near zero indicates that reaction operates close to equilibrium so near-equilibrium reactions are considered to be freely reversible since a slight excess of products can easily drive the reaction in reverse] ~(3) phosphofructokinase- consumes ATP to phosphorylate fructose-6-phosphate to yield fructose-1,6-biphosphate [when glycolytic pathway is producing plenty of phosphoenolpyruvate and ATP the phosphoenolpyruvate can act as feedback inhibitor to slow pathway by decreasing rate of reaction catalyzed by phosphofructokinase] [phosphofructokinase reaction is primary control point for glycolysis since its the slowest reaction and the rate largely determines the flux of glucose through entire pathway]

peptide bonds

~peptide bonds can be broken/hydrolyzed by exo or endopeptidases (enzymes that act from end or middle of chain respectively) ~chain of amino acid residues linked by peptide bonds is written so residue w/ free amino group is on left (N terminus) and free carboxylate group is on the right (C terminus) [except for 2 terminal groups the charged amino and carboxylate groups of each amino acid are eliminated in forming peptide bonds so properties of polypeptide therefore depend on identities of side chains] ~chemical properties of side chains immediate neighbors (microenvironment) may alter its polarity thereby altering its tendency to lose/accept proton ~for polypeptide of 100 residues there are 20^100 possible amino acid sequences

proteins (2)

~polar amino acids have side chains that can react w/ water b/c they contain H bonding groups [Ser, Thr, Tyr have hydroxyl groups, Cys has tiol group, Asn and Gln have amide groups, His has imidazole ring] [all of these can be found on solvent exposed surface of protein] [Gly has only H as side chain so cant H bond but it included w/ polar amino acids b/c its neither hydrophobic nor charged] [depending on presence of nearby side groups that increase polarity some of the polar side chains can ionize at physiological pH values] ~4 amino acids have side chains that are always charged under physiological condition [Asp and Glu have carboxylate groups that are negatively charged and Lys and Arg are positively charged] [side chains are located on proteins surface where they can be surrounded by water molecules or interact w/ other polar or ionic substances]

tertiary structure

~polypeptide segment that has folded into single structural unit w/ hydrophobic core is called domain [some proteins consist of single domain while others contain several which may be structurally similar or different] ~core of domain is rich in regular secondary structure b/c α helices and β sheets which are internally H bonded minimize hydrophilicity of polar backbone groups [irregular secondary structures are found on surface where polar backbone groups can form H bonds to water molecules] ~the largest force governing protein structure is the hydrophobic effect which causes nonpolar groups to aggregate [this stabilizes folded polypeptide backbone since unfolding it would expose hydrophobic side chains to solvent] [H bonding by itself isnt major determinant of protein stability b/c in unfolded protein polar groups could just as easily form H bond w/ water molecules]

hydrophobic effect

~readily hydrated substances are hydrophilic but compounds that lack polar groups are hydrophobic [its thermodynamically unfavored to dissolve hydrophobic substance in water because when hydrophobic molecules is hydrated it becomes surrounded by layer of water molecules that cant paritcipate in normal H bonding so align themselves so that polar ends arent oriented toward nonpolar solute] [this constraint represents loss of entropy b/c of formation of cage of water molecules] ~when large number of nonpolar molecules are introduced they dont become individually hydrated they clump together (oil droplets) removing themselves from contact w/ water [entropy of nonpolar molecules is reduced but this is offset by ability of water molecules to interact freely] ~hydrophobic effect- exclusion of nonpolar substances from aqueous solution [aggregate only b/c they are driven out of aqueous phase by unfavorable entropy cost of individually hydrating them]

denaturation of DNA

~stability of DNA depends mostly on stacking interactions which are form of van der waals interaction b/w adjacent base pairs [although individual stacking interactions are weak they are additive along length of DNA molecule] ~stacking interactions b/w neighboring G-C base pairs are stronger than those of A-T base pairs (this isnt related to fact that G-C have one more H bond) [DNA helix that is rich in GC is harder to disrupt] ~midpoint of melting curve (Tm) is the temp where half the DNA has separated into single strands ~rate of renaturation of denatured DNA depends on length of double stranded molecule [short segments anneal faster b/c bases in each strand must locate partners along length of complementary strand]

monosaccharides (2)

~the numerous hydroxyl groups allow multiple points for chemical reactions to occur [one is the intramolecular rearrangement where carbonyl group reacts w/ one of its hydroxyl groups to form cyclic structure] [convert Fischer to Haworth projection by groups projecting to right in fischer will point down in haworth and groups projecting to left will point up] ~cyclization may either cause α anomer (hydroxyl group is on opposite side of ring from CH₂OH group that determines D or L configuration) or β anomer (hydroxyl group is on same side of ring as CH₂OH) [unlike enantiomers and epimers which are not interchangeable anomers in aqueous solution interconvert b/w α and β forms unless hydroxyl group attached to anomeric carbon is linked to another molecule] [solution of glucose is 64% β anomer, 36% α anomer and trace amounts of open chain form] ~hexoses and pentoses that undergo cyclization do not actually form planar structures (as they are drawn in haworth) but forms chair (so each C can retain its tetrahedral bonding geometry) [substituents of each carbon may point above (axial) or outward (equatorial)] [glucose can form chair where all bulky substituents occupy equatorial positions and this is probably why (stability) glucose is so abundant] [all other hexoses have some groups that are axial and so are less stable]

PCR

~uses DNA polymerase to make copies of particular DNA sequence [reaction mixture contains DNA sample, DNA polymerase, all 4 dNTPs, 2 oligonucleotide primers that are complementary to the 3' ends of the 2 strands of each target sequence] ~(1) sample is heated to 90-95 degrees C to separate DNA strands (2) lowered to 55 C so primers hybridize w/ DNA strands (3) increased to 75 C and polymerase synthesizes new DNA strands by extending primers [this is repeated as many as 40 times and sequence doubles in concentration w/ each reaction cycle (so exponential growth)] ~use bacterial DNA polymerase (Taq DNA polymerase) b/c they can withstand high temps required for strand separation ~qPCR- PCR continually generates new DNA sequences that bind to fluorescent probes so amount of DNA can be monitored over time rather than at the end of many reaction cycles

Km

~when [S]=Km the reaction velocity (v₀) is equal to half its maximum value (v₀=Vmax/2) [since Km is the substrate concentration at which reaction velocity is half maximal it indicates how efficiently an enzyme selects its substrate and converts it to product] ~the lower the Km the more effective the enzyme is at low substrate concentrations and the higher Km the less effective it is [Km is often used as measure of enzymes affinity for substrate (approximates dissociation constant of ES complex) (Km=[E][S]/[ES])]

bilayer/micelles

~when molecules have nonpolar and polar portions they are amphipathic [when these are added to water the polar groups orient themselves toward solvent molecules and are hydrated while nonpolar groups tend to aggregate due to hydrophobic effect so form spherical micelle w/ solvated surface and hydrophobic core] ~bilayer is where hydrophobic layer is sandwiched b/w hydrated polar surfaces ~to eliminate solvent exposed edges of bilayer they tend to close up to form vesicle [when it forms it traps part of solution and polar solutes remain there b/c they cant easily pass through hydrophobic interior of vesicle] [normally substances that are present at high concentrations tend to diffuse to regions of lower concentrations but barrier like bilayer can prevent this diffusion]

acid base chemistry (3)

~when pH=pKa then acid is half dissociated [when pH<pKa the acid exists mostly in [HA] form and when pH>pKa acid exists mostly in [A⁻] form] ~many functional groups on biological molecules act as acids/bases and ionization states depend on pH of environment [pKa of carboxylic acid is 4 and of amine is 10 so when pH<4 its COOH and NH₃⁺, when 4<pH<10 its COO⁻ and NH₃⁺, when pH>10 then its COO⁻ and NH₂] ~buffers- when strong acid is added to pure water the acid added contributes directly to decrease in pH but when added to solution of weak acid in equilibrium w/ its conjugate base the pH doesnt change as dramatically [b/c some added protons combine w/ conjugate base to reform acid] [HCl→H⁺+Cl⁻ (large increase in [H⁺])] [HCl+A⁻→HA+Cl⁻ (small increase in [H⁺]) ~weak acid/base conjugate system acts as buffer against added acid or base by preventing dramatic changes in pH [pH doesnt change dramatically w/ added acid or base when pH is near pKa (effective buffering capacity of acid is within one pH unit of its pKa)] ~le Chatliers principle- change in concentration of one reactant will shift concentrations of other reactants in order to restore equilibrium ~acid base balance in humans- many metabolic processes generate acids which must be buffered so they dont cause blood to drop below normal pH of 7.4 [functional groups can serve as buffers but most imp involves CO₂] [CO₂+H₂O↔H₂CO₃↔H⁺+HCO₃⁻]

enzyme inhibition (2)

~where substrate analogs make good competitive inhibitors transition state analogs make even better inhibitors b/c in order to catalyze reaction the enzyme must bind to the reactions transition state ~noncompetitive inhibition- inhibitor binds to site on enzyme other than active site that elicits conformational change that affects structure or chemical properties of active site [as a result kcat and Vmax decrease but Km doesnt change] ~mixed inhibition- inhibitor binds in way that both Vmax and Km are affected [Km may either increase or decrease] ~uncompetitive inhibitors- in multisubstrate reaction inhibitor can bind to enzyme after one substrate has bound to prevent reaction from continuing and yielding product [Vmax is lowered and Km is lowered to same degree] [increasing substrate concentration reverses effects of competitive inhibitors but does nothing for mixed, noncompetitive or uncompetitive inhibition]

aqueous chemistry

~why do drugs contain fluorine- F can take place of H in structure w/o significantly altering shape and has high electronegativity so behaves more like O than H [C-F is also electron withdrawing group which decreases basicity of nearby amino groups so it allows drug to move more easily through membranes to enter cells] [polar C-F bond can participate in H bonding so it increases the attraction b/w drug and target molecule] ~water has high dielectric constant which is measure of solvent ability to diminish electrostatic attractions b/w dissolved ions [higher the dielectric constant of solvent the less able ions are to associate w/ each other] ~inside cell spaces b/w molecules may only be wide enough for 2 water molecules to fit so this allows solute molecules to slide past each other and keep molecules from coming into van der waals contact (maintains crowded but fluid state)