Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition

Transcription

1 Inhibition of Enzyme Activity Types of Inhibition: ompetitive oncompetitive Uncompetitive Product Inhibition Suicide Inhibition ompetitive Inhibition Fig 815 1

2 ompetitive Inhibition MPETITIVE c structrually resembles S, but is not an S c binds to free E at active site where S binds c competes with S for free E igh S overcomes inhibition because all E is bound in ES complex; since rate [ES] and [ES] is max, rate is max; no EI c is present K c Equilibria Scheme E S ES P E K m Ic EI c slope = K m. (1 [I c ] / K c ) / V max c I c V o I c yint = 1/V max c S o 1 / S o xint = 1/K m slope = K m /V max yint = 1 / (K m. (1 [I c ] / K c )) oncompetitive Inhibition Fig 815 2

3 oncompetitive Inhibition MPETITIVE nc is not structurally similar to S; is not an S nc binds to free E or ES at a site where S does not bind nc does T compete with S for free E igh S cannot overcome inhibition because I nc binds to ES complex, inactivating it Equilibria Scheme E S ES P E K m I nc I nc K nc K' nc EI nc S ESI nc (inactive) V o I nc I nc slope = K m. (1 [I nc ] / K c ) / V max I So yint = (1[I nc ] / K nc ) / V max xint = 1/K m 1 / S o slope = K m /V max yint = 1/V max Examples: ompetitive and oncompetitive Inhibition **AD (reduced form) pyruvate **AD 2 3 Llactate DEED BI BI MEAISM AD PY LA AD S 1 S 2 P 1 P 2 E ES 1 ES 1 S 2 EP 1 P 2 EP 2 E 3

4 Examples: ompetitive and oncompetitive Inhibition LATATE DEYDGEASE **AD (reduced form) pyruvate **AD Llactate IIBITS: oxamate ethyloxamate AD For multisubstrate rxns, the type of inhibition depends upon the substrate that is varied in the inhibition experiment! LATATE DEYDGEASE **AD (reduced form) pyruvate **AD Llactate IIBITS: 2 What substrate does this inhibitor resemble? AD AD 4

5 Which plot describes the inhibition of lactate dehydrogenase by AD when AD is the varied substrate? 2 **AD (reduced form) 3 2 pyruvate AD 2 **AD oncompetitive I Llactate ompetitive inhibition seen if varied S is AD ompetitive I DEED BI BI MEAISM AD PY LA AD S 1 S 2 P 1 P 2 E ES 1 ES 1 S 2 EP 1 P 2 EP 2 E 1/AD 1/AD 2 Which plot describes the inhibition of lactate dehydrogenase by AD when pyruvate is the varied substrate? **AD (reduced form) 2 3 pyruvate AD 2 **AD Llactate DEED BI BI MEAISM AD PY LA AD S 1 S 2 P 1 P 2 E ES 1 ES 1 S 2 EP 1 P 2 EP 2 E oncompetitive inhibition seen if varied S is pyruvate oncompetitive ompetitive I I 1/PYUVATE 1/PYUVATE 5

6 2 Which plot describes the inhibition of lactate dehydrogenase by oxamate when AD is the varied substrate? **AD (reduced form) 3 2 pyruvate 2 2 oxamate **AD Llactate oncompetitive inhibition seen if varied S is AD oncompetitive I DEED BI BI MEAISM AD PY LA AD S 1 S 2 P 1 P 2 E ES 1 ES 1 S 2 EP 1 P 2 EP 2 E ompetitive I 1/AD 1/AD Which plot describes the inhibition of lactate dehydrogenase by oxamate when pyruvate is the varied substrate? 2 **AD (reduced form) 2 3 pyruvate 2 2 oxamate **AD oncompetitive I Llactate DEED BI BI MEAISM AD PY LA AD S 1 S 2 P 1 P 2 E ES 1 ES 1 S 2 EP 1 P 2 EP 2 E ompetitive inhibition seen if varied S is pyruvate ompetitive I 1/PYUVATE 1/PYUVATE 6

7 Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind UMPETITIVE Equilibria Scheme u is not structurally similar to S; is not an S u binds to ES only; S opens up a site for u I u binding site may be in active site but binding of I u requires prior binding of S igh S cannot overcome inhibition because presence of S is required to provide a site for binding of u I E S ES P E K m K u I u EI u yint = (1 [I u ] / K u ) / V max I u 1/V o I V o I u slope = K m / V max So xint = (1 [I u ] / K u ) / K m xint = 1 / K m 1 / S o yint = 1 / Vmax 7

8 Example: Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind **AD 2 IIBIT: GLYEALDEYDE3PSPATE DEYDGEASE 2 Pi glyceraldehyde3pi As P 2 **AD (reduce d form) DEED TI BI MEAISM P 2 Pi 1,3bisphosphoglycerate AD GAP Pi 1,3BPG AD S 1 S 2 S 3 P 1 P 2 E ES1 ES' 1 P 2 ES'1P2 EP 1 P E 2 EP 2. Predict the type of inhibition by 2 As 4 when each of the substrates is varied in inhibition experiments This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind **AD 2 IIBIT: GLYEALDEYDE3PSPATE DEYDGEASE 2 Pi glyceraldehyde3pi As P 2 **AD (reduce d form) DEED TI BI MEAISM P 2 Pi 1,3bisphosphoglycerate AD GAP Pi 1,3BPG AD S 1 S 2 S 3 P 1 P 2 E ES1 ES' 1 P 2 ES'1P2 EP 1 P E 2 EP 2. 8

9 Predict the type of inhibition by 2 As 4 when each of the substrates is varied in inhibition experiments DEED TI BI MEAISM Pi S 3 P AD GAP 1,3BPG AD S 1 S 2 P 1 P 2 As E ES 1 ES' 1P 2 ES' 1 P 2 EP1 P E 2 EP 2 oncompetitive I. ompetitive I Uncompetitive I 1/S o 1/S o If S o = Pi 1/S o If S o = AD or GAP Product Inhibition Equilibria Scheme PDUT IIBITI p is structurally similar to S E S K ES P E m p binds to free E at active site where S binds P p competes with S for free E K At low S, resembles competitive inhibition p owever, at high S, the inhibition is not overcome EI because higher levels of P are generated which p inhibit the enzyme V o slope = K m / V max S o 1 / S o xint = 1 / K m 9

10 Example: Product Inhibition bgalactosidase (lactase) lactose galactose glucose Suicide Inhibition This type of enzyme inhibition results in the stoichiometric covalent modification of a side chain on an amino acid in the active site of an enzyme. The inhibitor chemically resembles a (one of the) substrate(s) and binds in the active site in the same way as the substrate(s) binds. The inhibitor, however, has a functional group, ususally a leaving group, that is replaced by a nucleophile in the enzyme active site. This covalent enzymeinhibitor complex forms irreversibly, thereby irreversibly inactivating the enzyme. Therefore this type of inhibition is called "suicide inhibition" or affinity labeling and the inhibitor is called a "suicide inhibitor". This reaction with the suicide inhibitor removes active enzyme from the system; this removal is measured as inhibition. Since active enzyme is lost, the inhibition is not relieved at high substrate levels. The rate, at high substrate in the presence of the inhibitor,is still proportional to the amount of the enzymesubstrate complex. owever, the maximum amount of that complex is limited by the remaining amount of active enzyme, not by the total enzyme added to the system. V o I I S o 1 / S o MichaelisMenten and LineweaverBurk plots look noncompetitive 10

11 Suicide Inhibition I V o I S o 1 / S o k 1 k2 E S ES P E k 1 u: X ( = I) E X The suicide inhibitor removes E so that the [ES] is lower, V max is lower, and inhibition cannot be overcome at high S o u inactived enzyme Example: Suicide Inhibition with hymotrypsin ne synthetic substrate for chymotrypsin I hymotrypsin Inhibitor tosyl phenylalanyl chloromethylketone 1 / S o tpck 11

12 k 1 k 2 E S ES P E k 1 u: X ( = I) E u Example: Suicide Inhibition with hymotrypsin X inactived enzyme at all S o, V o % [ES] chemically resembles S (I added first, S second to start reaction) reacts 1:1 with enzyme usually lowers [E], therefore lowering [ES] and V o lowers maximum amount of [ES] because it removes E V o can never reach V max resembles noncompetitive inhibition because inhibition cannot be relieved at high S YS= : l 2 2 S 3 l (X) YS= irreversibly inactivated Suicide Inhibitors: Aspirin Drug 2 Target Enzymes (Box 211) 3 Aspirin yclooxygenases 1 and 2 aspirin 2 2 a ibuprofen "Simple" eversible Inhibitors 3 naproxen tylenol acetylcysteine antidote for tylenol poisoning S 12

13 yclooxygenase and Modification by Aspirin aspirin 2 3 ox1 2 2 salicylic acid ox Drug Suicide Inhibitors: ew SAIDs Target Enzyme elebrex Vioxx yclooxygenase 2 Tylenol and Vioxx, two other medications commonly used for arthritis, were similarly tested Both groups showed no competitive interaction with aspirin. Two phases of inhibition: 1. apid competitive inhibition 2. Slower irreversible inhibition (covalent modification) 13

14 Suicide Inhibitors: Inhibitors of monoamine oxidase (MA) for treatment of depression l l lorgyline Deprenyl Pargyline 14