Types of Inhibition: Competitive Noncompetitive Uncompetitive Product Inhibition Suicide Inhibition

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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 is bound in ES complex; since rate [ES] and Kc [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

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 of S is required to provide a site for binding ouf I EI u

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

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 because higher levels of P are generated which EIp inhibit the enzyme 1 / 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. 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 Vo +I -I

So 1 / So

Michaelis-Menten and Lineweaver-Burk plots “look” noncompetitive

10 Suicide Inhibition

+I 1 / Vo -I -I Vo +I

So 1 / So k1 k2 E + S ES P + E k -1 Nu: + R-X ( = I) The suicide inhibitor removes E so that the [ES] is lower, Vmax is lower, and inhibition X cannot be overcome at high S E o

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 k2 E + S ES 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 X -I lowers [E], therefore lowering [ES] and Vo E -I lowers maximum amount of [ES] because it removes E -Vo can never reach Vmax Nu -resembles noncompetitive inhibition because inhibition cannot be relieved at high S R inactived enzyme

O O

CHY-HIS=N : Cl CH2 C CH NH S CH3 CHY-HIS=N- R CH O 2 irreversibly Cl inactivated (X)

Suicide Inhibitors: Aspirin CO H Drug 2 Target Enzymes

(Box 21-1) O C C H 3 Aspirin Cyclooxygenases 1 and 2 aspirin O O

C C H 3 CO2H CO2Na CH NH CH 3 CH CH3

"Simple" Reversible Inhibitors OH CH2 O CH tylenol CH3 O CH3 CH3 - CH3CNHCHCO2 ibuprofen naproxen N-acetylcysteine CH2 antidote for SH http://cti.itc.virginia.edu/~cmg/Demo/pdb/cycox/cycox_2.html tylenol poisoning

12 Cyclooxygenase and Modification by Aspirin http://www.chem.uwec.edu /Webpapers_F98/bruce/Pages/aspcommands .html

CO2H + Cox-1 O C C H 3 CH2OH aspirin O

CO H 2 Cox-1 + OH CH2O

salicylic acid C C H 3 O

http://www.scripps .edu /pub/goodsell/pdb/pdb17/pdb17_1.html

Suicide Inhibitors: New NSAIDs Drug Target Enzyme Celebrex 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

Pargyline N

http://www.csusm.edu/DandB/AD.html