1. ENZYMES KINETICS, INHIBITION, REGULATION
Muhammad Jawad Hassan
Assistant Professor
Biochemistry

2. Michaelis-Menten kinetics
V0 varies with [S]
Vmax
approached
asymptotically
V0 is moles of product
formed per sec. when [P]
is low (close to zero time)
E + SESE + P
Michaelis-Menten Model
V0 = Vmax x[S]/([S] + Km)
Michaelis-Menten Equation

3. Steady-state & pre-steady-state conditions
At pre-steady-state,
[P] is low (close to zero
time), hence, V0 for
initial reaction velocity
At equilibrium,
no net change of [S] & [P]
or of [ES] & [E]
At pre-steady state, we can ignore the back reactions

4. Michaelis-Menten kinetics (summary)
Enzyme kinetics (Michaelis-Menten Graph) :
At fixed concentration of enzyme, V0 is almost linearly proportional
to [S] when [S] is small, but is nearly independent of [S] when [S]
is large k1 k2
Proposed Model: E + S  ES  E + P
ES complex is a necessary intermediate
Objective: find an expression that relates rate of
catalysis to the concentrations of S & E, and the rates
of individual steps

5. Michaelis-Menten kinetics (summary)
Start with: V0 = k2[ES], and derive,
V0 = Vmax x[S]/([S] + Km)
At low [S] ([S] < Km), V0 = (Vmax/Km)[S]
At high [S] ([S] > Km), V0 = Vmax
When [S] = Km, V0 = Vmax/2.
Thus, Km = substrate concentration at which
the reaction rate (V0) is half max.

6. Range of Km values
Km provides approximation of [S] in vivo for many enzymes

7. Lineweaver-Burk plot (double-reciprocal)

8. Allosteric enzyme kinetics
Sigmoidal dependence of V0 on [S], not Michaelis-Menten
Enzymes have multiple subunits
and multiple active sites
Substrate binding may be cooperative

9. Enzyme inhibition

10. A competitive inhibitor

11. Methotrexate
A competitive inhibitor of dihydrofolate reductase - role in purine
& pyrimidine biosynthesis
Used to treat cancer

12. Kinetics of competitive inhibitor
Increase [S] to
overcome
inhibition
Vmax attainable,
Km is increased
Ki =
dissociation
constant for
inhibitor

13. Competitive inhibitor
Vmax unaltered, Km increased

14. Kinetics of non-competitive inhibitor
Increasing [S] cannot
overcome inhibition
Less E available,
Vmax is lower,
Km remains the same
for available E

15. Noncompetitive inhibitor
Km unaltered, Vmax decreased

16. Enzyme inhibition by DIPF
Group - specific reagents react with R groups of amino acids
diisopropylphosphofluoridate
DIPF (nerve gas) reacts with Ser in acetylcholinesterase

17. Affinity inhibitor: covalent modification

18. Catalytic strategies commonly employed
Covalent catalysis. The active site contains a reactive group, usually a nucleophile that becomes temporarily covalently modified in the course of catalysis
2. General acid-base catalysis. A chemical reaction is catalyzed by an acid or a base. The acid is often the proton and the base is often a hydroxyl ion. A molecule other than H2O may play the role of a proton donor or acceptor.

19. 3. Metal ion catalysis. Metal ion can function in several ways;
• can serve as an electrophile, stabilizing a negative charge on a reaction intermediate.
• can generate a nucleophile by increasing the acidity of a nearby molecule, such as H2O in the hydration of CO2 by carbonic anhydrase.
• can bind to substrate, increasing the number of interactions with the enzyme.
4. Catalysis by approximation. Bringing two substrates together along a single binding surface on an enzyme

20. Enzyme specificity: chymotrypsin
Cleaves proteins on carboxyl side of aromatic, or large hydrophobic amino acid
Bonds cleaved, indicated in red
The enzyme needs to generate a powerful nucleophile to cleave the bond

21. A highly reactive serine (#195) in chymotrypsin
27 other serines not reactive to DIPF,
Ser 195 is a powerful nucleophile
DIPF: di-isopropylphosphofluoridate, only reacts with Ser 195

22. Covalent catalysis Hydrolysis in two stages Deacylation to regenerate
free enzyme
Acylation to form
acyl-enzyme intermediate
Ser 195 OH group
attacks the carbonyl group
Acyl-enzyme intermediate
is hydrolysed

23. Chymotrypsin in 3D See Structural Insights
3 chains; orange,
blue, & green
Catalytic triad of
residues, including
Ser 195
2 interstrand, &
2 intrastrand
disulfide bonds
See Structural Insights
Synthesized as chymotrypsinogen
Proteolytic cleavage to 3 chains

24. The catalytic triad (constellation of residues)
Ser 195 converted into a potent nucleophile, an alkoxide ion
Asp 102
orients
His 57
Imidazole N as
base catalyst,
accepts H ion,
positions &
polarizes Ser
H ion withdrawal from
Ser 195 generates
alkoxide ion

25. Regulatory Strategies: Enzymes & Hemoglobin
Allosteric control. Proteins contain distinct regulatory sites and multiple functional sites. Binding of regulatory molecules triggers conformational changes that affect the active sites.
Display cooperativity: small [S] changes - major activity changes. Information transducers: signal changes activity or information shared by sites
2. Multiple forms of enzymes (isozymes). Used at distinct locations or times. Differ slightly in structure, in Km & Vmax values, and in regulatory properties
3. Reversible covalent modification. Activities altered by covalent attachment of modifying group, mostly a phosphoryl group
4. Protleolytic activation. Irreversible conversion of an inactive form (zymogen) to an active enzyme

26. Aspartate transcarbamoylase reaction
Committed step in pyrimidine synthesis: inhibited by end product
CTP

27. CTP inhibits ATCase

28. CTP stabilizes the T state
CTP binds to
regulatory
subunits

29. R and T states in equilibrium

30. ATCase displays sigmoidal kinetics
Substrate binding to one active site converts enzyme to R state
increasing their activity: active sites show cooperativity

31. Basis of sigmoidal curve
R & T states equivalent to 2 enzymes with different Kms
Cooperativity

32. Effect of CTP on ATCase kinetics
CTP stabilizes the T state, curve shifts to right

33. Effect of ATP on ATCase kinetics
ATP, allosteric activator, stabilizes R state, curve shifts to left

34. Oxygen delivery by hemoglobin, cooperativity enhanced
= 66%
= 38%
Cooperativity
enhances
delivery 1.7 fold
Partial pressure of oxygen

35. Heme group structure 4 linked pyrrole rings form a tetrapyrrole
ring with a central
iron atom.
side chains attached

36. Position of iron in deoxyhemoglobin
Iron slightly outside
porphyrin plane
His (imidazole ring)
binds 5th
coordination site
6th site for O2 binding

37. O2 binding, conformational change
Iron moves into plane, his is pulled along

38. Quaternary structure of hemoglobin
Pair of identical
alpha-beta dimers

39. Transition from T-to-R state in hemoglobin
Interface most affected
As O2 binds, top pair rotate 15o with respect to bottom pair

40. Oxygen affinity of fetal v maternal red blood cells
Fetal Hgl does not
bind 2,3-BPG,
higher O2 affinity
Fetal hemoglobin
tetramer has
2 alpha & 2 gama
chains,
Gene duplication

41. Isozymes of lactate dehydrogenase: glucose metabolism
Rat heart LDH isozyme profile changes with development
H(heart) isozyme (chain)= square, M(muscle) isozyme = circle

42. Tissue content of LDH
Functional LDH is tetrameric, with different combinations of subunits possible.
H4 (heart) has higher affinity for substrates than does M4 isozyme,
different allosteric inhibition by pyruvate H4
H3M
H2M2
HM3 M4
Some isozymes in blood indicative of tissue damage, used for clinical diagnosis
Increase in serum levels of H4 relative to H3M, indicative of
myocardial infraction (heart attack)

43. Examples of covalent modification

44. Phosphorylation widely used for regulation
Gamma
phosphoryl
group

45. Some known protein kinases

46. Protein phosphotases
Reverse the effects of kinases, catalyze hydrolytic removal of
phosphoryl groups attached to proteins

47. Activation by proteolytic cleavage

48. Secretion of zymogens by acinar cell of pancreas
Pancreas, one of the most
active organs in
synthesizing & secreting
proteins
Acinar cell stimulated by
hormonal signal or
nerve impulse, granule
content released into
duct to duodenum

49. Proteolytic activation of chymotrypsinogen
Active enzyme generated
by cleavage of a single
specific peptide bond
3 chains linked by 2
interchain disulfide
bonds, (A-B & B-C)

50. Conformations of chymotrypsinogen & chymotrypsin
Electrostatic interaction
between Asp 194
carboxylate & Ile 16
-amino group possible
only in chymotrypsin,
essential for activity

51. Zymogen activation by proteolytic cleavage
Zymogens orange, active enzymes yellow
Secreted by cells
that line duodenum
Digestive proteins of duodenum

52. Interaction of trypsin with its inhibitor
Lys 15 & Asp 189
form salt bridge
inside the active
site

53. Thank You