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Biochemistry BBMB 301

by: Emily

Biochemistry BBMB 301 BBMB 301


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Week 3 Lecture Notes
Survey of Biochemistry
Robert Thornburg
Class Notes
biochemistry, BBMB
25 ?




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This 7 page Class Notes was uploaded by Emily on Tuesday February 9, 2016. The Class Notes belongs to BBMB 301 at Iowa State University taught by Robert Thornburg in Spring 2016. Since its upload, it has received 9 views. For similar materials see Survey of Biochemistry in General Science at Iowa State University.


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Date Created: 02/09/16
Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 6 – CHAPTER 6 – CONCEPTS OF ENZYME ACTION By: Emily Settle  Classes of Enzymes o Oxidoreductases – REDOX enzymes transfer electrons between compounds o Transferases – transfer functional groups between compounds o Hydrolases – adds (or removes) water to another compound o Lyases – adds (or removes) functional groups to a double bond o Isomerases – moves functional groups to another spot in a compound o Ligases – joins two molecules but costs an ATP molecule  Reaction rate o Number of molecules of product formed per second, or o Number of molecules of reactant lost per second o Rate is proportional to the concentration of reactant or reactants o Rate of forward reaction is proportional to amount of CO2 present  The more CO2 present, the more H2CO3 is formed  Enzymes are catalysts – catalysts do not change the equilibrium of the reaction, but it does change the rate of the chemical reaction o Enzymes affect reaction rates o Enzymes do not affect equilibrium points  Ex  hydrolysis of a peptide bond o The equilibrium of this reaction favors hydrolysis o The rate of extremely slow in the absence of catalyst, so proteins are stable o Specific peptide hydrolyzing enzymes increase the rate many fold  Catalyzed vs. Non-catalyzed reaction rates o Gibbs free energy, ΔG  Energy of activation  Transition state energy  Factor that considers chemical energy changes and entropy changes o Enzymes reduce transition state energy  Thus they increase the reaction rate  Orient reactants productively  Occurs by binding of reactants to enzyme in active site  Negative change in G for a reaction, -ΔG o more energy in reactants than in products o Increase in the enthalpy (randomness) after the reaction is complete o spontaneous  Positive change in G for a reaction, +ΔG o less energy in reactants than in products o decrease in the enthalpy (randomness) after the reaction is complete o not spontaneous  Enzyme activated reactions: energy does not change ΔG, but Energy of activation is reduced.  Consider a reaction starting at arbitrarily defined standard conditions o A + B   C + D  The particular ΔG at these conditions is called ΔG°’  Enzymatic Reactions o Absence of catalyst: Substrate (S)  Product (P) Keq = [P]/[S] o Enzyme-catalyzed reaction: E + S  ES  EP  E + P  Reduces to S  P Keq = [P]/[S]  Enzymes do not affect Keq  They affect the rate at which P is formed o Binding of S to E changes the reaction environment  “easier” for S to convert to P  In other words, the frequency of passing the transition state is increased  Because, the transition state energy level is reduced when E binds to S  Active sites – substrate binds to enzyme in the active site o Localized region in the tertiary structure of the enzyme o Binding occurs owing to close-fitting weak bonds between the enzyme and the substrate o Close fit of bonds necessary to bind substrate imparts specificity to the enzyme o The active site usually is within an exposed cleft in the enzyme  The active site residues usually are from distant regions of primary sequence  Brought together in space by protein folding  Non-catalytic water molecules usually excluded  Models of active site action o Lock & Key model  The enzyme has a defined structure and  The substrate has a similarly defined structure  The two components fit together uniquely o Induced fit model  The enzyme molds itself to the substrate, getting tighter as the substrate is converted into the transition state  This occurs owing to small changes in protein conformation  Cofactors o Active sites often require non-amino acid components, called cofactors o Two classes  Metal ions  Small organic molecules called coenzymes o Chemical nature of the cofactors is involved in reduction of activation energy o Protein lacking the cofactor is called the apoenzyme Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 7 – CHAPTER 7 – ENZYME KINETICS AND REGULATION By: Emily Settle  Study enzyme structure o X-ray crystallography o NMR o Protein sequencing  Study enzyme action o Identify substrates o Synthesis of products o Reaction parameters  How fast it works  Substrate binding  Enzyme Assay o V = change in product with time (Δ[P]/ Δt) o Or change in substrate with time (Δ[S]/ Δt)  Absorbance spectroscopy o S or P absorbs photons of a given wavelength o Absorbance change proportional to moles S or P o Convert mathematically to µmol/min  Radioactive labeling o S or P gain or lose a radioactive atom o Separate labeled species, measure counts per minute  Scintillation counter o Values proportional to moles of S or P  Enzyme assays can help define substrates and products and can begin to evaluate activity.  Enzyme Reaction Parameters o How fast will it work?  Maximum velocity – Vmax o How tightly does it bind to the substrate  Km = Michaelis-Menton Constant  Michaelis-Menton Equation – describes the enzyme activity (V0) as a function of substrate concentration [S]  Lineweaver-Burke Plot – inverse of the initial velocity vs. the inverse of the substrate concentration  Meaning of Km – indicates how well E binds to S o High Km = E has a low affinity for S o Low Km = E has high affinity for S o Affinity measures proportion of time that S spends bound to E  This depends entirely on molecular structure  Results from weak bonds between S and E  Meaning of Vmax – shows how quickly E catalyzes conversion S to P o AFTER S has bound to E o This value determined by activation energy  A related value is k2 (also kcat) o This is the turnover number o Moles S converted to P by one enzyme molecule in 1 second o Units are moles/second, or s^-1  Multiple Substrates o Investigate order of binding  Determine Km for S1 and S2 individually  Hold concentration of one substrate constant, vary the other to determine K o Possible binding orders  Random sequential  Either A or B can bind first  Either P or Q can leave first  Ordered sequential  A must bind before B can bind  A must leave before B can leave  Michaelis-Menton Enzymes vs. Allosteric Enzymes o M-M enzymes are always “on”  The rate of reactions they catalyze depend only on how much substrate is present o Allosteric enzymes are sometimes “on” and sometimes “off”  When off, the rates of the reactions they catalyze are not increased when [S] increases  Regulation is mediated by small organic molecules  These bind at a place different from the active site and change the enzyme structure to increase or decrease activity  Allosteric Regulation – Effect of Regulatory Molecules o Regulatory molecules affect the position of the equilibrium  This changes the efficiency of the enzyme at a given substrate concentration  Regulators can increase the activity accelerating the reaction rate  Or they can decrease the activity thereby decelerating the reaction rate  Quaternary structure participates in allosteric mechanisms  Feedback regulation o Enzymes often work in sequential pathways  Product of one enzyme is substrate for the next  Full pathway regulated by activity at one step  Usually an early step  Feedback regulation stops metabolic waste  Reversible o Coordinates complex pathways  Stimulation  Inhibition  Adjusts levels of substrates to insure equivalent levels of intermediates  Enzyme Regulation o Genetic control  Which enzymes are present, what concentration? o Compartmentalization – keep substrate and enzyme apart o Covalent modification  Activation or repression  Addition/removal of functional groups, cleavage of certain peptide bonds o Allosteric regulation  Small molecules (metabolites) affect enzyme activity  Metabolic pathways are reprogrammed  Fine tuning of enzyme activity, rapid response  Regulatory enzymes are regulated, complex network – feedback regulation  Metabolic networks o Controls required so that pathways with opposite functions do not operate at the same time o Functions of key enzymes in pathways are modulated Biochemistry 301 – Survey of Biochemistry Professor Robert Thornburg LECTURE 8 – CHAPTER 8 – ENZYME MECHANISMS AND INHIBITORS By: Emily Settle  Covalent catalysis – covalent modification of the enzyme during the mechanism  General acid-base catalysis – a molecule functions as a proton donor or acceptor o Active site groups serve as acids or bases  NH3+, -COOH, -COO-, -NH2, His imidazole  Donate or accept H+ to stabilize intermediate  Acid- and base- catalysis differ in the step that happens first  Acid catalysis – proton donated to form intermediate  Base catalysis – proton abstracted to form intermediate  Metal ion catalysis – metal ions bound to the enzyme function in the mechanism o Metal ions associated with an enzyme or substrate often participate in catalysis o Common metal ions – Na+, K+, Mg+2, Mn+2, Cu+2, Zn+2, Fe+2, Fe+3, Ni+2 o Metal ions assist catalysis in three ways – orient substrate in active site by coordination o Enhance a reaction by polarizing the scissle bond or stabilizing a negatively charged intermediate o REDOX reaction, accepting and donating electrons  Metals serve as electron carriers  Proximity effects – two substrates are brought into close proximity in the mechanism  Nucleophilic functional group on enzyme forms a covalent bond with the substrate o A highly reactive transient intermediate form is produced o The covalent bond to the enzyme subsequently is broken to form the product  Mechanisms of Enzymatic Catalysis o Proximity and strain effects  Substrates held in place  Vibrational and kinetic energy captured  Structural change in enzyme places strain on substances  Energy must be applied, brings substrate toward transition state o Electrostatic effects  Substrates de-solvate when they bind enzyme  Ex  water shell is removed  Charge distribution on the substrate is destabilized  Increases reactivity  Water molecules typically are excluded from the active site  Temperature enhances the rate of Enzyme-Catalyzed Reactions o Optimal temperature for many human enzymes is at 37 degrees Celsius o Thermophilic bacteria exist at a much higher temperature and the enzymes show temperature optima reflect this o Ectothermic (cold-blooded) organisms absorb heat from the environment to adjust body temperature so that enzymes will work more efficiently.  Effects of pH on Enzyme Action – most enzymes have an optimal pH o Most enzymes show a bell-shaped curve surrounding the pH optimum where the enzyme exists o Pepsin is a stomach enzyme which is active at about pH 2.0 (pH of stomach juice) o Chymotrypsin is expressed in the intestine where the pH is closer to 8.0  Enzyme Inhibition o Organic molecules often reduce enzyme activity  Specific inhibitors for specific enzymes o Importance  Regulation of metabolism and biological activity  Enzymes in the cell are turned on and off by inhibitors  Study of reaction mechanisms, enzyme function  Clinical therapies – angiogenesis inhibitors  Toxins - proteinase inhibitors  Types of Inhibitors o Irreversible inhibitors (suicide inhibitors)  Usually covalent, enzyme permanently inactivated  Suicide inhibition destroys the inhibitor o Reversible inhibitors  Non-covalent, inhibitor can dissociate then activity restored  Three types  Competitive – binds active site, competes with substrate  Noncompetitive – inhibitor binds regulatory site, distant from active site, disables the catalytic mechanism  Uncompetitive – similar to noncompetitive but inhibitor binds only to ES complex, not empty E  Inhibitors can be distinguished via their kinetics o Experiment  Determine Km and Vmax as usual  Constant [E], vary [S], measure Vo, plot Vo as a function of [S]  Repeat presence of a constant [l]  Vary [l] value  Evaluate via Lineweaver-Burke plot (+/-) inhibitor  Substrate mimics as competitive inhibitor o Inhibitor structure reveals functional groups on substrate that are important for interacting with the active site in the enzyme  Transition state analogs are good competitive inhibitors because they bind more tightly to enzyme than the substrate does  Serine Protease Mechanism (Chymotrypsin) o Reaction is to hydrolyze a peptide bond o Enzyme is specific for hydrolysis of the peptide bond following certain R groups  Aromatic – Trp, Phe  Large hdyrophobics – Met, Val, Ile  Chymotrypsin Protease Mechanism o Amino acid arrangement in enzyme makes serine hydroxyl a strong nucleophile  Catalytic triad: Asp-His-Ser o A synthetic substrate that releases a colored product is used for kinetic analysis  Product can be detected and quantified o Step 1 – rapid release of one of the products o Step 2 – slow release of second product


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