Enzyme Inhibition:
Very important way to control enzymes
Many drugs work in this manner- by reducing or blocking enzyme activity
Irreversible inhibition
Inhibitor I binds to E and permanently blocks activity
ASA (Aspirin) Irreversible Inhibitor
Acetylsalicylic acid
acetylates active site Serine in enzyme COX- Kills COX activity (cyclooxygenase)
Reversible Inhibition:
Advantage-enzyme is not permanently inactivated
Not a covalent modification
If [I] ↑ get inhibition
If [I] ↓ E activity ↑
a) Competitive Inhibition
Here I looks like S, binds to E at active site but cannot undergo reaction.
e.g. succinate dehydrogenase (SDH)
COOCOO
CH2
HC
CH2
↔
CH
COOCOO+ FAD
+ FADH2
Malonate: Competitive Inhibitor
COOCH2
COOMalonate
(3C) cannot react
Thus malonate binding to E slows reaction
Competitive I shifts vo vs [S] curve to the right
Vmax unchanged, but Km is increased (Km*) in presence of I
as S binding ↓in presence of I
Lineweaver Burk plot shows decrease in 1/Km* intercept
b)Non Competitive Inhibition
Binding site for I is distinct from active site.
E can bind S and I Thus I binding will not interfere with S binding
vo vs [S] plot shows no change in Km in presence of non-competitive I
But non-competitive I does lower Vmax = decreased catalytic efficiency
Likely binding of I causes conformational change in enzyme
For non-competitive inhibition
Lineweaver-Burk Plot shows larger 1/Vmax* than 1/Vmax
(Vmax*< Vmax)
Allosteric Regulation
commonly seen with multisubunit enzymes
ATCase (aspartate transcarbamoylase)
very important in synthesis of pyrimidine nucleotides:
CTP: cytidine triphosphate
UTP: uridine triphosphate
ATCase (E. coli) 310kD
12 subunits, forms carbamoyl aspartate
ATCase = 1st step in enzyme pathway to CTP and UTP
CTP, UTP inhibit ATCase (feedback inhibition)
However, ATP activates ATCase
CTP, ATP are allosteric modulators of ATCase
ATCase kinetics: Sigmoid plot of vo vs [Aspartate]
Sigmoid curve indicates cooperativity
CTP shifts curve to right:
ATP shifts curve to left: more like hyperbola
Allosteric kinetics very different from M-M kinetics
Apparent Km for Asp appears to
↑ in presence of CTP and
↓ in presence of ATP
High [CTP] shuts down CTP synthesis.
High [ATP] = signal for cell division
Structure of ATCase
6 catalytic subunits c and
6 regulatory subunits r: c6r6
r binds ATP or CTP
c carries out reaction
c gives a hyperbolic plot (no regulation if its by itself)
r: has no catalytic activity but does bind ATP or CTP
Enzyme Mechanisms
Ribonuclease A (RNase A) 14 kDa, 124 amino acids,
4 -S-S- bridges (4 cystines)
RNase A= small pancreatic enzyme
RNase A attaches to long polymeric RNA and cuts the chain
Investigations of nature of active site:
1. Incubate RNase A at 3oC briefly with bacterial protease subtilisin
One peptide bond hydrolysed between AA20 and 21
Enzyme product = RNase S and it was active.
RNase S did hang together (non-covalent forces)
When two pieces pulled apart no activity in S-peptide (AA 1-20) or S-protein (21-124)
Each part contributed to the active site of the enzyme
2. Chemical Modification:
Incubate RNase A with iodoacetate: loss of activity
This inactive RNase A has carboxymethyl His 12 or carboxymethyl His 119.
Suggested His 12 and 119 are at the active site and play a role in catalysis.
3. RNase A cuts RNA on the 3’ side of pyrimidine nucleotide residues (C or U)
Active site of RNase A may have a binding site for the pyrimidine nucleotides.
Mechanism: RNase A
Acid-Base catalysis where
His 12, 119 serve as H+ donors and acceptors
Note mechanism in figure.
Chymotrypsin (CT):
CT also a pancreatic enzyme, 25 kDa
Member of a family of enzymes called serine proteases (trypsin, elastase, subtilisin, thrombin)
Each member: same catalytic mechanism. Each has same “catalytic triad” at active site.
CT: triad
Asp 102..His 57..Ser 195
Ser does not normally lose a H+ from its –OH (no dissociation)
However close proximity of His strongly attracts H so that Ser can dissociate.
Asp stabilizes +ve charge on His
See Figure for mechanism.
Overall: The triad allows for a peptide bond in a protein or peptide to be broken.
A new N- and C-terminus are generated-the protein is broken into 2 smaller pieces
What distinguishes different members of the Serine protease family if they all have the
same mechanism?
Answer: Binding specificity
Each member: a different binding pocket promotes hydrolysis of different peptide bonds
Enz. Pocket Specificity
CT H-phobic Trp, Tyr, Phe
T -ve charge Lys, Arg
El. Short Ala, Gly
Each cuts on carboxyl side of the amino acid listed.