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 K -Ic binds to free E at active site where S binds + m -I competes with S for free E c Ic -High S overcomes inhibition because all E K is bound in ES complex; since rate [ES] and c [ES] is max, rate is max; no EIc is present EI c slope = Km . (1+ [I c] / K c) / Vmax +I c -Ic 1 / Vo +Ic -I Vo y-int = 1/Vmax c So 1 / So x-int = -1/Km slope = Km/Vmax yint = -1 / (Km . (1+ [I c ] / Kc )) Noncompetitive Inhibition Fig 8-15 2 Noncompetitive Inhibition NONCOMPETITIVE Equilibria Scheme -Inc is not structurally similar to S; is not an S E + S ES P + E + Km -Inc binds to free E or ES at a site where S + does not bind I nc I nc -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 (inactive) - Inc slope = K m . (1+ [In c ] / Kc ) / Vmax +I + Inc 1 / V Vo o -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+ O H O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **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 O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **NAD (oxidized form) HO H O O O C NH - - INHIBITORS: 2 H2N C CO2 CH3CH2HN C CO2 N 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 O H H O OH C - C + CH C CO - H2N + CH3 C CO2 NH2 3 2 H N N pyruvate L-lactate R R **NADH (reduced form) **NAD (oxidized form) HO H O C What substrate does this INHIBITORS: NH2 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 ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R O **NADH HO H E ES ES S EP P EP2 E (reduced form) **NAD 1 1 2 1 2 C (oxidized form) NH2 N Competitive inhibition R NAD-OH seen if varied S is NADH Noncompetitive Competitive +I +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 ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R O **NADH HO H E ES ES S EP P EP2 E (reduced form) **NAD 1 1 2 1 2 C (oxidized form) NH2 N Noncompetitive inhibition R NAD-OH seen if varied S is pyruvate Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/PYRUVATE 1/PYRUVATE 5 Which plot describes the inhibition of lactate dehydrogenase by oxamate when NADH is the varied substrate? + H ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R **NADH O EP **NAD E ES1 ES1S2 EP1P2 2 E (reduced form) - (oxidized form) H2N C CO2 oxamate Noncompetitive inhibition seen if varied S is NADH Noncompetitive Competitive +I +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 ORDERED BI BI MECHANISM O H H H O + O OH NADH PYR LAC NAD H N C + - C + CH C CO - 2 CH3 C CO2 NH2 3 2 S1 S2 P1 P2 H N N pyruvate L-lactate R R **NADH O EP **NAD E ES1 ES1S2 EP1P2 2 E (reduced form) - (oxidized form) H2N C CO2 oxamate Competitive inhibition seen if varied S is pyruvate Noncompetitive Competitive +I +I 1 / Vo 1 / Vo -I -I 1/PYRUVATE 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 UNCOMPETITIVE Equilibria Scheme -Iu is not structurally similar to S; is not an S E + S ES P + E Km -Iu binds to ES only; S opens up a site four I + -Iu binding site may be in active site but binding Iu of I u requires prior binding of S K -High S cannot overcome inhibition because presence u u of S is required to provide a site for binding of I EIu y-int = (1+ [Iu] / Ku ) / V max + Iu +I 1/Vo - Iu -I Vo slope = Km / Vmax So 1 / So x-int = -(1+ [Iu] / Ku ) / Km y-int = 1 / Vmax x-int = -1 / Km 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 O O H H O C C H O - NH C O P O 2 + - C - H C OH + HO P O H2N O + H C OH N OH CH2OPi N CH2OPi R glyceraldehyde-3-Pi R 1,3-bisphosphoglycerate **NAD **NADH (oxid ized fo rm ) (red uce d fo rm) O INHIBITOR: HO As O- ORDERED TRI BI MECHANISM OH NAD + G AP Pi 1,3- BPG N ADH S1 S2 S3 P1 P2 E ES1 ES'1P2 ES'1P2 E EP1P2 EP2 . - Predict the type of inhibition by H2AsO4 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 O O H H O C C H O - NH C O P O 2 + - C - H C OH + HO P O H2N O + H C OH N OH CH2OPi N CH2OPi R glyceraldehyde-3-Pi R 1,3-bisphosphoglycerate **NAD **NADH (oxid ized fo rm ) (red uce d fo rm) O INHIBITOR: HO As O- ORDERED TRI BI MECHANISM OH NAD + G AP Pi 1,3- BPG N ADH S1 S2 S3 P1 P2 E ES1 ES'1P2 ES'1P2 E EP1P2 EP2 . 8 - Predict the type of inhibition by H2AsO4 when each of the substrates is varied in inhibition experiments ORDERED TRI BI MECHANISM O + GAP Pi O 1,3-BPG NADH NAD HO AsO- HO P O- P P S1 S2 S3 1 2 OH OH ES'1P2 ES' P E E ES1 1 2 EP1P2 EP2 . Noncompetitive Competitive Uncompetitive +I +I 1 / Vo 1 / Vo 1 / Vo +I -I -I -I 1/So 1/S o 1/S o If So = Pi If So = NADH or GAP Product Inhibition Equilibria Scheme PRODUCT INHIBITION E + S ES P + E K -Ip is structurally similar to S + m -Ip binds to free E at active site where S binds P -Ip competes with S for free E -At low S, resembles competitive inhibition Kp -However, at high S, the inhibition is not overcome EI because higher levels of P are generated which p inhibit the enzyme 1 / Vo Vo slope = Km / Vmax So 1 / So x-int = -1 / Km 9 Example: Product Inhibition b-galactosidase (lactase) HOCH lactose 2 O HOCH2 OH OH HO O O OH OH O OH H H HOCH2 HOCH2 HO O OH O OH OH HO OH OH OH 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 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.