1. ways to determine Km and Vmax
1.1. Km=substrate concentration that yields Vmax/2
1.2. Vmax is only approachable but never attained
1.3. most commonly achieved with the use of curve fitting programmes on a computer
1.4. older method=Lineweaver-Burk plot
1.4.1. rarely used because data points at high and low cons. are weighted differently
1.4.1.1. sensitive to errors
1.4.2. impossible to obtain a definitive and accurate value of KM and Vmax
1.4.3. involves use of algebraic manipulation to determine Vmax and KM
1.4.3.1. Vmax can be accurately determined if MM eqn is transformed into an eqn that gives a straight line plot
1.4.3.2. take reciprocal of both sides
1.4.3.2.1. resembles formula for straight line graph
1.4.3.2.2. y=mx+c
1.4.3.3. used to produce a plot of 1/V0 vs 1/[S]
1.4.3.3.1. called Lineweaver-Burk or double-reciprocal plot
1.4.3.3.2. yields a straight line with a
2. Enzyme inhibition
2.1. activity of many enzymes can be inhibited by binding of specific small molecules/ions
2.2. inhibitors
2.2.1. molecules that prevent enzymes from carrying out their normal function
2.2.2. serve as major control mechanisms in biological systems
2.2.2.1. e.g. regulation of allosteric enzymes
2.2.3. many enzymes are regulated by inhibitors
2.2.4. many drugs and toxic agents can serve as inhibitors
2.2.4.1. some enzyme inhibitors are impt. medicinal drugs
2.2.4.1.1. should understand how inhibitors work
2.3. inhibition can be a source of insight into the mechanism of enzyme action
2.3.1. specific inhibitors can be used to identify residues critical for catalysis
2.3.2. e.g. transition state analogs are especially potent inhibitors
2.4. 2 types of enzyme inhibition
2.4.1. irreversible inhibition
2.4.1.1. irreversible inhibitor dissociates very slowly from its target enzyme
2.4.1.2. the inhibitor becomes tightly bound to the enzyme
2.4.1.2.1. covalently/non covalently
2.4.1.3. some irreversible inhibitors are impt. drugs
2.4.1.3.1. penicilllin
2.4.1.3.2. aspirin
2.4.1.4. 3 categories
2.4.1.4.1. group specific reagents
2.4.1.4.2. reactive substrate analogs/affinity labels
2.4.1.4.3. suicide inhibitors
2.4.1.5. 2 ways functional grps required 4 enzymatic activity are determines
2.4.1.5.1. X-ray crystallography
2.4.1.5.2. irreversible inhibitors
2.4.2. reversible inhibition
2.4.2.1. characterized by rapid dissociation of enzyme-inhibitor complex
2.4.2.1.1. can bind to enzyme and dissociate easily
2.4.2.2. competitive inhibition
2.4.2.2.1. one type of reversible inhibition
2.4.2.2.2. enzyme can bind substrate>ES complex or it can bind to an inhibitor>EI complex
2.4.2.2.3. competitive inhibitor often resembles the substrate and binds to the active site of the enzyme
2.4.2.2.4. competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate
2.4.2.2.5. substrate dependent
2.4.2.2.6. Vmax can still be reached
2.4.2.2.7. increasing Km of enzyme
2.4.2.2.8. example of competitive inhibitors
2.5. Non-competitive inhibition
2.5.1. inhibitor and substrate bind to the enzyme at the same time but on different binding sites
2.5.2. inhibitor binds to diff site on enzyme
2.5.2.1. causes conformational change in enzyme
2.5.2.1.1. substrate can no longer bind
2.5.3. can bind a free enzyme or an ES complex
2.5.4. acts by decreasing the concentration of functional enzyme
2.5.4.1. net effect=decrease in turnover number
2.5.5. cannot be overcome by increase in substrate conc.
2.5.5.1. substrate independent
2.5.5.2. doesnt affect KM
2.5.6. e.g. of mixed inhibition
2.5.6.1. when a single inhibitor both hinders the binding of a substrate and decreases the turnover # of the enzyme
2.5.7. inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites
2.6. Uncompetitive inhibition
2.6.1. inhibitor binds only to the ES complex
2.6.2. uncompetitive inhibitor's binding site is only created on the interaction of the E and S
2.6.3. cannot be overcome by addition of more substrate
2.6.3.1. substrate independent
2.6.4. KM decreases
2.6.4.1. less [ES] complexes formed
2.6.4.1.1. less substrate and enzyme formed
3. Penicillin
3.1. first antibiotic discovered
3.2. e.g. of a clinically used suicide inhibitor
3.2.1. suicide substrate
3.2.2. rxn kills the enzyme
3.3. MOA
3.3.1. inhibits glycopeptide transpeptidase which cross links bacterial cell walls
3.3.2. has similar structure to normal substrate and binds to active site on enzyme
3.4. structure
3.4.1. consists of
3.4.1.1. thiazolidine ring fused to a beta-Lactam ring
3.4.1.2. a variable R grp is attached to the beta-Lactam ring by a peptide bond
3.4.1.2.1. beta-Lactam ring is v. labile
3.4.1.2.2. variable grp in benzylpenicillin=benzyl grp
3.4.2. structure can undergo a variety of rearrangements
3.5. how it inhibits bacterial growth
3.5.1. e.g. S. aureus
3.5.1.1. most common cause of staph infections
3.5.1.2. penicillin interferes with synthesis of cell wallls
3.5.1.3. cell wall is made up of peptidoglycan (macromolecule)
3.5.1.3.1. consists of linear cross linked polysaccharide chains
3.5.1.3.2. peptidoglycan cell wall gives mechanical support to the bacterial cells and prevents them from bursting in response to high internal osmotic pressure
3.5.1.4. Glycopeptide transpeptidase
3.5.1.4.1. catalyzes the formation of cross links that make peptidoglycan stable
3.5.1.4.2. bacterial cell walls contain D-amino acdis
3.5.1.5. penicillin inhibits cross linking by transpeptidase by the Trojan horse stratagem
3.5.1.5.1. transpeptidase normally forms an acyl intermediate with penultimate D-alanine residue of D-Ala-D-Ala peptide
3.5.1.5.2. penicillin mimics D-Ala-D-Ala part of the substrate
3.5.1.5.3. bound penicillin forms a covalent bond with a serine residue at the AS of the enzyme
3.5.1.5.4. penicilloyl-enzyme does not react further
3.6. why is it an effective inhibitor
3.6.1. four membered highly strained B-lactam ring
3.6.1.1. especially reactive
3.6.2. when penicillin binds to transpeptidase
3.6.2.1. serine residue at AS of enzyme attacks the carbonyl carbon atom of the lactam ring
3.6.2.1.1. form penicilloyl-serine derivative
3.6.3. suicide inhibitor
3.6.3.1. transpeptidase participates in activation of it
4. distinguishing reversible inhibitors
4.1. consider only enzymes that exhibit MM kinetics
4.2. by measuring the rates of catalysis at diff. concentrations of substrate and inhibitor we can distinguish the 3 types of inhibition
4.3. can distinguish between
4.3.1. competitive inhibition
4.3.1.1. inhibitor competes with the substrate for the active site
4.3.1.2. dissociation constant for the inhibitor Ki
4.3.1.2.1. Ki=[E][I]/[EI]
4.3.1.2.2. smaller Ki=more potent inhibition
4.3.1.2.3. new value of KM=KMapp
4.3.1.3. main feature
4.3.1.3.1. can be overcome by a sufficiently high conc. of substrate
4.3.1.4. increase in inhibitor=increases the value of KM
4.3.1.4.1. more substrate is needed to obtain the same rate of rxn
4.3.1.5. reaction pathway
4.3.1.5.1. high concs. of substrate can completely relieve competition inhibition
4.3.1.6. Vmax stays the same in its presence
4.3.1.6.1. virtually all ASs can still be filled at sufficiently high[S]
4.3.1.6.2. enzyme is fully functional
4.3.1.7. commonly used as drugs
4.3.1.7.1. iboprofen
4.3.1.7.2. statins
4.3.2. non competitive inhibition
4.3.2.1. substrate can still bind to EI complex
4.3.2.1.1. EIS complex does not form the product
4.3.2.2. Vmax is decreased to new value Vappmax
4.3.2.2.1. given by
4.3.2.3. KM is unchanged
4.3.2.3.1. inhibitor simply lowers the concentration of functional enzyme
4.3.2.3.2. resulting solution acts like a dilute enzyme
4.3.2.3.3. non competitive inhibition cannot be overcome by increasing the substrate concentration
4.3.2.4. rxn pathway
4.3.2.4.1. inhibitor binds both to free enzyme and to ES complex
4.3.2.4.2. Vmax cannot be attained
4.3.2.4.3. Km remains unchanged
4.3.2.4.4. rxn rate increases more slowly at low substrate concs.
4.3.2.5. e.g.
4.3.2.5.1. deoxycycline (antibiotic)
4.3.2.5.2. lead
4.3.3. uncompetitive inhibition
4.3.3.1. inhibitor binds only to the ES complex
4.3.3.2. the ESI does not go on to form any product
4.3.3.2.1. unproductive ESI complex will always be present
4.3.3.3. Vmax will be lower in the presence of inhibitor
4.3.3.3.1. Vmax cannot be attained even at higher substrate concs.
4.3.3.4. inhibitor lowers the value of KM
4.3.3.4.1. inhibitor binds to ES>ESI
4.3.3.4.2. depleting ES
4.3.3.4.3. more S binds to E to maintain the equilibrium between E and ES
4.3.3.4.4. lower S conc. is needed to form half the maximal conc. of ES
4.3.3.4.5. becomes more smaller as inhibitor is added
4.3.3.5. e.g. the herbicide glyphosate/Roundup
4.3.3.5.1. uncompetitive inhibitor
4.3.3.5.2. inhibits the an enzyme in the biosynthetic pathway of aromatic AAs
4.3.3.6. rxn pathway
4.3.3.6.1. shoes that the inhibitor binds to only the ES complex
5. Lineweaver plot and inhibitors
5.1. double reciprocal plots can be used to distinguish between
5.1.1. competitive
5.1.1.1. intercept on y-axis of 1/V vs 1/[S] plot stays the same
5.1.1.1.1. 1/Vmax
5.1.1.1.2. inhibitor doesnt alter Vmax
5.1.1.2. slope is increased
5.1.1.2.1. KM/Vmax
5.1.1.2.2. indicates strength of binding of competitive inhibitor
5.1.1.2.3. slope plot increased by a factor of (1+[I]/Ki)
5.1.1.3. presence of competitive inhibitor halfs the rxn rate at a given substrate conc.
5.1.2. uncompetitive
5.1.2.1. inhibitor combines with the ES complex only
5.1.2.2. slope of line stays the same
5.1.2.2.1. KM/Vmax
5.1.2.3. y-axis intercept increases
5.1.2.3.1. 1/Vmax
5.1.2.3.2. by a factor of (1+[I]/Ki)
5.1.2.4. KM and Vmax reduced by equivalent amounts
5.1.3. non competitive inhibitors
5.1.3.1. inhibitor can combine to enzyme/ES complex
5.1.3.2. pure non competitive inhibition
5.1.3.2.1. value of dissociation constants of EI and EIS are =
5.1.3.2.2. Vmax decreases
5.1.3.2.3. new slope=KM/Vappmax
5.1.3.3. Vmax decreases,KM not affected
5.2. used to see effects of inhibitor
5.3. set up controls with and without the inhibitor
6. Transition-state analogs
6.1. potent inhibitors of enzymes
6.2. Linus Pauling 1928
6.2.1. proposed that compounds resembling the transition state of a catalyzed rxn should be very effective enzyme inhibitors
6.2.2. =transition state analogs
6.3. enzymes work by stabilizing the transition state
6.4. TS analogs work by binding tightly to the AS
6.5. highly potent and specific inhibitors of enzymes can be produced by synthesizing compounds that more closely resemble the TS than the substrate itself
6.6. Power of TS analogs
6.6.1. they are sources of insight into catalytic mechanisms
6.6.2. serve as potent and specific inhibitors of enzymes
6.6.3. can be used as antigens to generate a wide range of new catalysts
6.7. e.g. Proline biosynthesis
6.7.1. conversion of L-proline to D-proline requires proline racemase
6.7.2. racemization of proline proceeds through a TS in which the tetrahedral alpha C atom has become trigonal
6.7.2.1. trigonal form
6.7.2.1.1. all 3 bonds are in same plane
6.7.2.1.2. Calpha carries -ve charge
6.7.3. Pyrrole 2- carboxylic acid= TS analog has trigonal geometry
6.7.3.1. potent inhibitor of proline racemase
6.7.3.2. binds more tightly than proline
6.7.3.3. Alpha carbon atom of the inhibitor is also trigonal
6.8. Catalytic antibodies
6.8.1. demonstrate the importance of selective binding of the TS to enzymatic activity
6.8.2. a rxn is catalyzed by stabilizing the transition state
6.8.3. antibodies which recognize the TS should function as catalysts
6.8.4. e.g. preparation of an antibody that catalyzes the insertion of a metal ion into a porphyrin
6.8.4.1. Ferrochelatase catalyzes the insertion of Fe into the porphyrin ring of protoporphyrin IX
6.8.4.1.1. the planar porphyrin must be bent 4 iron to enter
6.8.4.1.2. proceeds via bent porphyrin transition state
6.8.4.2. N-MethylMesoporphyrin=a potent inhibitor of ferrochelatase
6.8.4.2.1. resembles the TS
6.8.4.2.2. N-alkylation forces force the porphyrin ring to bend
6.8.4.2.3. used to catalyze ferrochelatase rxn and generate an antibody
6.8.5. catalytic antibodies(abzymes) can be produced using TS analogs as antigens
7. Enzyme regulation
7.1. enzymes are often regulated
7.2. 2 types of regulation
7.2.1. allosteric
7.2.1.1. involves product inhibition
7.2.1.2. used to control flux through metabolic pathways
7.2.1.2.1. e.g. -ve feedback
7.2.1.3. allosteric enzymes dont show MM kinetics
7.2.1.3.1. have multiple subunits and many active sites
7.2.1.4. multimeric enzymes often have sigmoidal kinetics
7.2.1.4.1. while MM kinetics is hyperbolic
7.2.2. cooperative
7.2.2.1. binding of an enzyme to one binding site helps binding to other active sites
7.2.2.2. e.g. oxygen binding to Haemoglobin
8. Summary
8.1. Lineweaver-Burk plot can be used to determine Vmax and KM
8.2. inhibitors=substances that inhibit enzymes from working
8.3. inhibitors can be competitive,noncompetitive or non competitive
8.4. TS analogs can be used as competitive inhibitors of enzymes
8.5. enzymes need to be regulated-regulation is either allosteric/cooperative
8.6. Enzyme
8.6.1. without enzymes biological rxns wont take place
8.6.2. without inhibitors and enzyme regulation side and unwanted rxns would take place