Inhibition of Enzyme Activity

Inhibition of Enzyme Activity Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition Competitive Inhibition Fig 8-15 1 Competitive Inhibition COMPETITIVE Equilibria Scheme -Ic structrually resembles S, but is not an S E + S ES P + E Km -Ic binds to free E at active site where S binds + -Ic competes with S for free E Ic -High S overcomes inhibition because all E is bound in ES complex; since rate [ES] and Kc [ES] is max, rate is max; no EI c is present EI c slope = K m . (1+ [I c ] / K c ) / Vma x +Ic Vo +I c 1 / Vo -I c -I c y-int = 1/Vmax So 1 / So x-int = -1/Km slope = K m /Vmax yint = -1 / (Km . (1+ [I c ] / K c )) Noncompetitive Inhibition Fig 8-15 2 Noncompetitive Inhibition NONCOMPETITIVE Equilibria Scheme E + S ES is not structurally similar to S; is not an S Km + binds to free E or ES at a site where S + I nc I nc does not bind -Inc does NOT compete with S for free E K nc K'nc -High S cannot overcome inhibition because Inc binds to ES complex, inactivating it EI nc + S ESI nc P + E -Inc -Inc (inactive) - I nc slope = Km . (1+ [Inc ] / Kc ) / Vmax +I + I nc 1 / Vo Vo -I So 1 / So y-int = (1+[I nc] / Knc ) / Vmax slope = K m /Vmax x-int = -1/Km y-int = 1/Vmax Examples: Competitive and Noncompetitive Inhibition H+ H2N O H H C + O H O C CO2- CH3 C N OH + CH3 C CO2H NH2 N L-lactate pyruvate R **NADH (reduced form) R **NAD (oxidized form) ORDERED BI BI MECHANISM + NADH PYR LAC NAD S1 S2 P1 P2 E ES1 ES1S2 EP1P2 EP2 E 3 Examples: Competitive and Noncompetitive Inhibition LACTATE DEHYDROGENASE + H O H H H2N C H O + CH3 C C CO2- N INHIBITORS: NH2 N pyruvate R **NADH (reduced form) O + CH3 OH C CO2H L-lactate R **NAD (oxidized form) HO H O C NH 2 N O H2N C CO2- O CH3CH2HN C CO2- oxamate ethyloxamate R NAD-OH For multisubstrate rxns, the type of inhibition depends upon the substrate that is varied in the inhibition experiment! LACTATE DEHYDROGENASE H+ O H H H2N C H O + CH3 C pyruvate R **NADH (reduced form) INHIBITORS: C CO2- N O NH2 N OH + CH3 C CO2H L-lactate R **NAD (oxidized form) HO H O C NH 2 What substrate does this inhibitor resemble? N R NAD-OH NADH 4 Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when NADH is the varied substrate? H+ O H H HNC + 2 H O CH3 C CO2- C N ORDERED BI BI MECHANISM O NH2 N HO H O C (reduced form) NADH PYR LAC NAD+ S1 S2 P1 P2 L-lactate pyruvate R **NADH OH + CH3 C CO2H R E **NAD ES1 ES1S2 (oxidized form) EP2 EP1P2 E NH2 N Competitive inhibition seen if varied S is NADH R NAD-OH Competitive +I Noncompetitive +I 1 / Vo 1 / Vo -I -I 1/NADH 1/NADH Which plot describes the inhibition of lactate dehydrogenase by NAD-OH when pyruvate is the varied substrate? H+ O H H HNC + 2 O CH3 C CO2- N H ORDERED BI BI MECHANISM O C NH2 N (reduced form) HO H O C N R NAD-OH NADH PYR LAC NAD+ S1 S2 P1 P2 L-lactate pyruvate R **NADH OH + CH3 C CO2H R E **NAD (oxidized form) ES1 ES1S2 EP2 EP1P2 E NH2 Noncompetitive inhibition seen if varied S is pyruvate Noncompetitive Competitive +I 1 / Vo 1 / Vo -I 1/PYRUVATE +I -I 1/PYRUVATE 5 Which plot describes the inhibition of lactate dehydrogenase by oxamate when NADH is the varied substrate? H+ O H H HNC + 2 H O CH3 C CO2- C N ORDERED BI BI MECHANISM O NH2 N PYR LAC NAD+ S1 S2 P1 P2 R O (reduced form) NADH L-lactate pyruvate R **NADH OH + CH3 C CO2H H2N C CO2oxamate ES1 E **NAD ES1S2 (oxidized form) EP2 EP1P2 E Noncompetitive inhibition seen if varied S is NADH Competitive +I Noncompetitive +I 1 / Vo 1 / Vo -I -I 1/NADH 1/NADH Which plot describes the inhibition of lactate dehydrogenase by oxamate when pyruvate is the varied substrate? H+ O H H HNC + 2 O CH3 C CO2- N H ORDERED BI BI MECHANISM O C NH2 N O (reduced form) H2N C CO2- NADH PYR LAC NAD+ S1 S2 P1 P2 L-lactate pyruvate R **NADH OH + CH3 C CO2H R E **NAD (oxidized form) ES1 ES1S2 EP2 EP1P2 E oxamate Competitive inhibition seen if varied S is pyruvate Noncompetitive Competitive +I 1 / Vo 1 / Vo -I 1/PYRUVATE +I -I 1/PYRUVATE 6 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 Equilibria Scheme UNCOMPETITIVE -Iu -Iu -Iu is not structurally similar to S; is not an S E + S ES Km binds to ES only; S opens up a site for u I + binding site may be in active site but binding Iu of Iu requires prior binding of S Ku -High S cannot overcome inhibition because presence uI of S is required to provide a site for binding of EIu P+ E y-int = (1+ [Iu] / Ku ) / V max + Iu 1/Vo +I -I - Iu Vo slope = Km / Vmax 1 / So So x-int = -(1+ [Iu] / Ku ) / Km x-int = -1 / Km y-int = 1 / Vmax 7 Example: Uncompetitive Inhibition This type of inhibition requires that one or more substrates bind to E before the inhibitor can bind GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE H O O C NH2 N R **NAD O H H H2N C O C H + H C OH + HO P OOH CH2OPi R **NADH (oxid ized fo rm ) HO As OOH C O P-O O H C OH CH2OPi 1,3-b isphospho glycer ate (red uce d fo rm) O INHIBITOR: + N g lyceraldeh yd e-3-Pi O O ORDERED TRI BI MECHANISM NAD + GAP Pi E ES 1 N ADH 1,3-BPG P1 S1 S2 S3 ES' 1 P 2 ES' 1 P 2 P2 E EP 2 EP 1 P 2 . Predict the type of inhibition by H2 AsO4 - 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 GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE H O O C NH2 N R **NAD O H H H2N C O C H + H C OH + HO P OOH CH2OPi R **NADH (oxid ized fo rm ) HO As OOH C O P-O O H C OH CH2OPi 1,3-b isphospho glycer ate (red uce d fo rm) O INHIBITOR: + N g lyceraldeh yd e-3-Pi O O ORDERED TRI BI MECHANISM NAD + GAP Pi 1,3-BPG P1 S1 S2 S3 E ES 1 ES' 1 P 2 ES' 1 P 2 EP 1 P 2 N ADH P2 EP 2 E . 8 Predict the type of inhibition by H2 AsO4 - when each of the substrates is varied in inhibition experiments ORDERED TRI BI MECHANISM NAD + GAP O Pi S1 S 2 S 3 O NADH 1,3-BPG P1 HO P O- HO As O- P2 OH OH E ES'1P2 ES1 ES' 1P2 EP1P2 EP2 E . Competitive +I Noncompetitive +I Uncompetitive 1 / Vo 1 / Vo 1 / Vo -I +I -I -I 1/S o 1/So If So = Pi 1/S o If So = NADH or GAP Product Inhibition Equilibria Scheme PRODUCT INHIBITION E + S ES Km -Ip is structurally similar to S + -Ip binds to free E at active site where S binds P -Ip competes with S for free E Kp -At low S, resembles competitive inhibition -However, at high S, the inhibition is not overcome EIp because higher levels of P are generated which inhibit the enzyme 1 / Vo P+ E Vo slope = Km / Vmax So 1 / So x-int = -1 / Km 9 Example: Product Inhibition β -galactosidase (lactase) HOCH2 lactose O OH HOCH2 O HO OH O OH OH O OH H H HOCH2 HOCH2 O OH HO OH O OH OH HO OH OH glucose galactose 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 enzyme-inhibitor 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 enzyme-substrate complex. However, the maximum amount of that complex is limited by the remaining amount of active enzyme, not by the total enzyme added to the system. +I 1 / Vo -I +I Vo So -I 1 / So Michaelis-Menten and Lineweaver-Burk plots “look” noncompetitive 10 Suicide Inhibition +I 1 / Vo -I +I Vo -I So 1 / So k1 E + S ES k2 P + E k -1 Nu: + R-X ( = I) X E The suicide inhibitor removes E so that the [ES] is lower, Vmax is lower, and inhibition cannot be overcome at high So Nu R inactived enzyme Example: Suicide Inhibition with Chymotrypsin One synthetic substrate for chymotrypsin +I 1 / Vo -I Chymotrypsin Inhibitor tosyl phenylalanyl chloromethylketone 1 / So tpck 11 Example: Suicide Inhibition with Chymotrypsin k1 E + S ES k2 P + E k -1 Nu: + R-X ( = I) -at all So, Vo % [ES] -I chemically resembles S (I added first, S second to start reaction) -I reacts 1:1 with enzyme usually -I lowers [E], therefore lowering [ES] and Vo -I lowers maximum amount of [ES] because it removes E -V o can never reach Vmax -resembles noncompetitive inhibition because inhibition cannot be relieved at high S X E Nu R inactived enzyme O O Cl CH2 C CH NH S CHY-HIS=N : CH3 CHY-HIS=N- O CH2 Cl (X) R irreversibly inactivated Suicide Inhibitors: Aspirin CO2H Drug O C CH 3 Aspirin aspirin O CO2H CO2Na CH CH3 CH CH3 Target Enzymes (Box 21-1) Cyclooxygenases 1 and 2 O C CH 3 NH "Simple" Reversible Inhibitors CH2 CH CH3 CH3 ibuprofen O CH3 naproxen http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html OH tylenol O CH3 CNHCHCO2 CH2 N-acetylcysteine antidote for SH tylenol poisoning 12 Cyclooxygenase and Modification by Aspirin http://www.chem.uwec.edu/Webpapers_F98/bruce/Pages/aspcommands .html CO2H + Cox-1 O C CH 3 CH2OH aspirin O CO2H + Cox-1 OH salicylic acid CH2O C CH 3 O http://www.scripps.edu/pub/goodsell/pdb/pdb17/pdb17_1.html Suicide Inhibitors: New NSAIDs Drug Celebrex Target Enzyme Vioxx Cyclooxygenase 2 Tylenol and Vioxx, two other medications commonly used for arthritis, were similarly tested … Both groups showed no competitive interaction with aspirin. http://www.pslgroup.com/dg/1e8fa2.htm Two phases of inhibition: 1. Rapid competitive inhibition 2. Slower irreversible inhibition (covalent modification) 13 Suicide Inhibitors: Inhibitors of monoamine oxidase (MAO) for treatment of depression www.chem.vt.edu/chem-dept/office/jwolfe/DRUGCHEM1.ppt Cl Cl Clorgyline O N Deprenyl N Pargyline N http://www.csusm.edu/DandB/AD.html 14