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ENZYMES. Proteins that function as biological catalysts RNA catalysts are called ribozymes. S ↔ P . S = substrate(s) or reactant(s) & P = product(s). Enzyme Names S + type of reaction + ‘ ase ’ However, there are numerous exceptions.

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Enzymes

ENZYMES

Proteins that function as biological catalysts

RNA catalysts are called ribozymes

S ↔ P

S = substrate(s) or reactant(s) & P = product(s)

Enzyme Names

S + type of reaction + ‘ase’

However, there are numerous exceptions

Normal – hexokinase, lactate dehydrogenase

Abnormal – trypsin, DNA polymerase


Enzymes

  • catecholoxidase (1.10.3.1); pyruvatedehydrogenase (1.2.4.1)

  • hexokinase(EC 2.7.1.1); pyruvatekinase (2.7.1.40); RNA Polymerase (2.7.7.6)

3. carboxypeptidaseA (3.4.17.1); acetylcholinesterase (3.1.1.7)

4. carbonic anhydrase (4.2.1.1); adenylatecyclase (4.6.1.1)

5. phosphoglucoseisomerase (5.3.1.9); phosphoglucomutase (5.4.2.2)

6. DNA Ligase (6.5.1.1); acetyl CoA synthetase (6.2.1.1)


Enzymes

Assignment

Look up in Swiss Prot – UniprotKB database and Wikipedia

the following enzymes (human)

1) lactate dehydrogenase 4) carbonic anhydrase

2) glucokinase 5) phosphoglucomutase

3) carboxypeptidase A 6) DNA Ligase

Write ……

Name of enzyme & EC#

equation for reaction

Statement of its purpose/function of enzyme

http://www.expasy.org/



Enzymes

S  P

G = G+ RT ln ([P]/[S])

G = G

when all [ ]s = 1M

(except [H+] = 1 x 10-7M pH = 7.0)

At equilibrium G = 0

and

DGº' = -RT ln ([P]/[S])

G‡(activation energy)

determines the rate of the reaction

An Enzymeincreases the rate at which a reaction achieves equilibrium

by lowering the activation energy, Ea. It does not alter DGº′ nor change the final

equilibrium state.


Enzymes

G = G+ RT ln Q

where Q = ([P]/[S]) for the reaction S → P

A reaction is proceeding in a cell.

Which of the following best describes the free energy for this reaction.

a) DG is (–) and constant.

b) DG°′ must be (–).

c) DG is (–) and increasing.

d) None of the above


Enzymes

ES# ↔ EP

MichaelisMenten Model

k1

k2/kcat

E + S  ES  E + P

k-1

k-2

1 Enzyme (E) bind to substrate (S)

Lock & Key model

The Enzyme active site has a complementary shape to S

Weak bonds are formed between E & S

H-bonds, salt bridges, hydrophobic interactions & …

occasionally covalent bonds


Enzymes

Is shape the only factor that determines bonding

between E and S? a) yes b) no

Lock & Key Model

E & S have complementary shape

E & S form complementary interactions – H-bonds, salt bridges, and/or hydrophobic interactions



Enzymatic substrate selectivity
Enzymatic Substrate Selectivity

No binding

Binding but no reaction

Example: Phenylalanine hydroxylase


Enzymes

ES# ↔ EP

MichaelisMenten Model

k1

k2/kcat

E + S  ES  E + P

k-1

k-2

2 Enzyme converts S into P

The enzyme converts S into a conformation that looks like S#.

This requires some bonds in S to be weakened/stretched.

The enzyme positions multiple substrates so that new bonds

Can form when the ‘weakened’ bond(s) in S# breaks.


Enzymes

Initial rate assumption: [P] = 0 so k-2 [E] [P] = 0

Michaelis Menten Model

k1

k2

E + S  ES  E + P

k-1

k-2

0

rate of ES formation = k1 • [E] • [S] + k-2 • [E] • [P]

rate of ES dissappearance = k-1 • [ES] + k2[ES] = (k-1 + k2) [ES]

Steady state assumption – [ES] is constant

= k1 • [E] • [S] = k-1 [ES] + k2 [ES] = (k-1 + k2) [ES]

v = Vmax [S]

(KM + [S])

KM = (k-1 + k2)/k1


Enzymes

Michaelis Menten Model

k1

k2

E + S  ES  E + P

k-1

k-2

v vs. [S] plot is hyperbolic

1/v = KM/Vmax•1/[S] + 1/Vmax

Vmax = 1/yint & KM = slope • Vmax = -1/xint

v = Vmax [S]

(KM + [S])


Enzymes

Vmax

KM

Carbonic Anhydrase v vs. [S]

HCO3- + H+→ CO2 + H2O

v

Experiment:

Set up reactions with constant [E], and varying [S]

Measure reaction rate (v) for each [S]

Plot v vs. [S] and 1/v vs. 1/[S]

[E] = 1 x 10-6 M

Vmax = 1 Ms-1

KM = 0.012 M

kcat = 1 x 106 s-1

[S]


Enzymes

1/v = KM/Vmax * 1/[S] + 1/Vmax

Double Reciprocal Plot

Carbonic Anhydrase

KM = -1/Xint = slope•Vmax

1/v

slope = KM/Vmax

Yint = 1/Vmax

Xint = -1/KM

1/[S]


Enzymes

KM is ...

(k-1 + k2)/k1 - combined rate constant

[S] at which v = Vmax/2

 KM = tighter binding

Vmax (M•s-1) is ....

v at Enzyme saturation: [ES] = [E]tot

= k2 • [E]tot

k2 (or kcat)called turnover # (s-1) independent of [E]

An efficient enzyme will have ….. ?

a) a large value for KM b) a large value for kcat c) both

Enzyme efficiency

[S] >> KM

kcat

Diffusion limited if kcat/KM

~ 1 x108 to 1 x 109

[S] << KM

kcat/KM


Enzyme efficiency is limited by diffusion k cat k m
Enzyme efficiency is limited by diffusion:kcat/KM

  • Can gain efficiency by having high velocity or affinity for substrate

    • Catalase vs. acetylcholinesterase


Enzymes

ENZYME INHIBITORS

Competitive Noncompetitive

I binds to active site

I binds elsewhere

I prevents ES  E+P

I prevents S binding

KM - Vmax same

Vmax - KM same

I structurally

similar to S

I not similar to S


Enzymes

Vmax is represented by which Letter?

Which letter represents KM?


Enzymes

Vmax is represented by which of the following?

a) Yint b) slope c) 1/Yint d) slope/KM


Enzymes



Enzymes

Dihydrofolic noncompetitive acid

Folic acid

An important vitamin in humans, dihydrofolate is synthesized from

Para-aminobenzoic acid (PABA) in bacteria. RDI = 400 mg/day.

(1000 in pregnant women).

Sources: Green leafy vegetables, fruits, nuts, beans, peas, dairy.

(particularly high in asparagus and Brussel sprouts.)


Enzymes

H noncompetitive2N- -COOH

BACTERIA PATHWAY

GTP

folK

folQ

folE

folC

folP

dihydrofolate

PABA

DHPS

DHFS

Glutamate

dihydropteroate

PABA


Enzymes

Sulfa Drugs noncompetitive

NH2

Icomp

S = PABA

NH2

C=O

|

OH

NH2

O=S=O

|

NH2

O=S=O

N-H

N S

sulfathiazole


Enzymes

Sulfthiazole noncompetitive resistance case study

1985 – 5 isolates of resistant StreptoccoccusPyogenessavedfrom

patients in Sweden Hospital

1990’s – Genomes from normal and resistant isolates compared

– highly mutated genes cloned & expressed in E. coli.

DHPS gene found to be mutated.

KMKi

DHPS Kinetics

G1 (suscep)0.7mM 0.2mM

G56 (res) 2.5mM 27.4mM

Difference 3.6x 137x

Which enzyme will have better evolutionary ‘fitness’ in the absence of Inhibitor?

a) susceptible b) resistant

The resistant enzyme is less viable in absence of inhibitor but natural selection

favors resistant form in presence of sulfa drugs.


Enzymes

Today’s topics: noncompetitive

Review Enzyme Inhibitor type

Carboxypeptidase Mechanism

Enzyme Regulation

What type if inhibitor is represented here?

a) competitive

b) noncompetitive

c) uncompetitive

Which of the following best describes this inhibitor?

a) It must have a similar structure to the substrate

b) It prevents the substrate from binding

c) It increases Vmax.

d) None of the above


Enzymes

I is structurally similar to S noncompetitive

binds active site

Prevents E + S ↔ ES

↑ KM Vmax same

I binds to a separate site

prevents ES → E + P

↓Vmax KM same

I binds neat active site

if S is already bound then prevents ES → E + P

if S is not bound then prevents E + S → ES

↓Vmax KMcould go up or down depending …


Enzymes

Tryptathionone noncompetitive (oxidized) + NADPH ↔ Tryptathionone(reduced) + NADP+

Noncompetitive Inhibition


Enzymes

Tryptathionone noncompetitive (oxidized) + NADH ↔ Tryptathionone(reduced) + NAD+

Noncompetitive Inhibition

Fig. 1: lineweaver-Burk plots showing the simple linear non-competitive inhibition of trypanothionereductase by TNQ2 either to NADPH (left, [trypanothione] constant at 100 µM) and trypanothione (right, [NADPH] constant at 200 µM); open circles, [TNQ2] = 0; closed circles, [TNQ2] = 1 µM; open squares, [TNQ2] = 2.5 µM; and closed squares, [TNQ2] = 5 µM


Enzymes

Alcohol Dehydrogenase un-competitive inhibition noncompetitive

NAD + alcohol ↔ aldehyde/ketone + NADH

Fig. 2. Inhibition of human ADH1C*2 by 5α-androstan-17β-ol-3-one (5α-dihydrotestosterone). The enzyme was assayed at 37 °C in 83 mM potassium phosphate, pH 7.3, 40 mMKCl and 0.25 mM EDTA buffer using a DU-7 spectrophotometer to measure the change in absorbance at 340 nm due to changing NADH concentrations (v=ΔA340 min−1). Duplicate assays were averaged. (A) The concentrations of 5α-androstan-17β-ol-3-one were 0 (●), 34 (■), 68 (▴) and 103 μM (♦) and the concentration of NAD+ was 0.5 mM. The data were fitted to the equation for uncompetitive inhibition, v=VA/(Km+A(1+I/Ki)), where V is the maximum velocity and A is the varied substrate. (B) The concentrations of 5α-androstan-17β-ol-3-one were 0 (●), 5 (■), 10 (▴) and 20 μM (♦) and the concentration of NADH was 0.1 mM. The data were fitted to the equation for competitive inhibition, v=VA/(Km(1+I/Ki)+A). The S.E. of the fits were <10%.


Carboxypeptidase a cpa
CARBOXYPEPTIDASE noncompetitive A (CPA)

Digestive Enzyme: Zn Peptidase Family ― 309 aa’s―MW = 34,760

(Polypeptide)n→ (polypeptide)n-1 + C-terminal amino acid

Example of Induced Fit

X-ray Structure w/wo Substrate

Mechanism Hypothesis

How does the substrate bind to the active site? (KM effects)

What features of this interaction contribute to the ‘turnover’? (Vmax effects)


Enzymes

H noncompetitiveH

| |

H3N+- C - C - N-CH-CH2- -OH

| || |

H O COO-

ARTIFICIAL SUBSTRATE = Gly-Tyr

CPA – Substrate Comparison


Enzymes

Atom Residue noncompetitive Position 

relative to Fe

C ARG127 -.01

C (CN3) ARG127+.43

C (CN3) ARG145+.50

C TYR248 +.50

OH TYR248+11.21

C GLU270 -.01

C (COO-) GLU270-1.64


Enzymes

Zn noncompetitive

Gly-Tyr


Enzymes

Zn noncompetitive

Gly-Tyr


Enzymes

Zn noncompetitive

Substrate

Y248


Enzymes

Y noncompetitive248-OH

HOH

HOH

HOH

HOH

HOH

HOH

HOH

E270

Zn +R127 +R145

H196 E72

H69


Enzymes

Y noncompetitive248

OH

H H

- CH-C-N-CH-CH2- O -OH

O COO-

E270

Zn +R127 +R145

H196 E72

H69

bonds contributing

to KM & binding

step.


Enzymes

Y noncompetitive248

Y248

OH

OH

H H

-CH-C-N-CH-CH2- O -OH

O COO-

E270

Zn +R127 +R145

H196 E72

H69

H-OH


Enzymes

Y noncompetitive248

OH

H H

-CH-C-N-CH-CH2- O -OH

O COO-

E270

Zn +R127 +R145

H196 E72

H69

H-OH


Enzymes

+ noncompetitive

H3N

E270

The amino end KM

but also Vmax since

H2O substrate not in

position.

Y248

OH

H H

CH-C-N-CH-CH2- O -OH

O COO-

Zn +R127 +R145

H196 E72

H69


Enzymes

CARBOXYPEPTIDASE A (CPA) noncompetitive

(Polypeptide)n→ (polypeptide)n-1 + C-terminal amino acid

  • CPA binds to S (a polypeptide) at the carboxy terminal end.

  • It prefers an aromatic or large nonpolar side chain. Binds to E NP pocket

  • The Zn coordinates to the C=O of S peptide bond (to be cut)

  • The carboxyl end of the molecule forms a salt bridge with Arg from E

  • Y248 – form H-bond with N-H of S peptide bond (to be cut)

  • The other substrate (H2O) forms 5th coordination site to Zn from E.

  • This is blocked by amino terminal end if S = dipeptide GY.

  • That is why GY is a poor substrate with tight binding (low KM) but low kcat.


Enzymes

O H noncompetitiveHH

|| | | |

- O - C- N- C - C - N-CH-CH2-

| || |

H O COO-

H H

| |

H3N+ - C - C - N-CH-CH2- -OH

| || |

H O COO-


Enzymes

H noncompetitiveH

| |

H3N+ - C - C - N-CH-CH2- -OH

| || |

H O COO-

In Glycyl-tyrosine the + amino terminal end of the dipeptide …

a) Forms a salt bridge with and Arg side chain from CPA.

b) Is the 5th ligand to the Zn2+ of CPA.

c) both of the above

d) none of the above

As a result of the interaction above Gly-Tyr has …..

a) A very small KM value.

b) A very small Vmax value.

c) both of the above

d) none of the above


Enzymes

An enzyme is …. noncompetitive

a) a protein

b) a catalyst

c) both of the above

d) none of the above

An enzyme will ….

a) increase the rate of a reaction

b) Increase the ratio of product to reactant at equilibrium

c) both of the above

d) none of the above


Enzymes

Metabolic Regulation noncompetitive

1. How Much Enzyme - Regulation of gene expression

2. Activity of Available Enzyme

Allosteric Enzymes – noncovalent binding of allosteric regulators

modifies the activity of certain emzymes.

Covalent Modifications

The addition of phosphate (most common), acetyl, etc. group

via covalent bonds can alter the activity of enzyme.

Proenzymes

A proenzyme (proprotein or zymogen for non-enzymatic proteins)

is inactive until a specific protease removes a peptide segment from

the polypeptide chain.


Enzymes

P noncompetitive1

E1

E2

P2

P3

E3

Metabolic Regulation

Rationale of Metabolic Regulation

S

The priority of what a cell will do with a particular substrate can be

modified to adapt to changing conditions within the cell.

How fast various enzymes react with a particular substrate regulates what

happens to the substrate (as opposed to thermodynamics).


Enzymes

Metabolic Regulation noncompetitive

2. Activity of Available Enzyme

Allosteric Enzymes – noncovalent binding of allosteric regulators

modifies the activity of certain emzymes.

Allosteric enzyme typically have …..

1) quaternary structure

2) an allosteric site to bind regulatory molecules

3) T (tense – less active) and R (relaxed – fully active) conformations

4) A sigmoidal v vs. [S] plot (they don’t follow MM kinetics)

Note the similarity between enzyme regulation (allosteric enzymes)

and the allosteric regulation of Hemoglobin.


Enzymes

V noncompetitivemax

Michaelis - Menten

Enzyme R

v

allosteric enzyme

T

[S]


Enzymes

V noncompetitivemax

v

allosteric enzyme

[S]

positive regulator

negative regulator


Noncovalent modification allosteric regulators
Noncovalent Modification: Allosteric Regulators noncompetitive

The kinetics of allosteric regulators differ from Michaelis-Menten kinetics.


Covalent modification
Covalent Modification noncompetitive

Enzyme has active & inactive forms

Transformation due to “phosphorylation” of Side Chain

Protein Kinase catalyzes Phosphoryl transfer (ATP donor)

Phosphatase reverses

OH

OPO3-

“Protein” kinase

+ ATP

“Protein” phosphatase

+ Pi

Result: “All of None” regulation of activity


Proenzymes
Proenzymes noncompetitive

Proenzyme synthesized and stored in inactive form

Transformation due to cleavage of “activation peptide”

A specific protease removes the activation peptide

Once activated the process is not reversible

Ultimately the active enzyme is hydrolyzed (as with all emzymes)

protease


Enzymes

TRYPSINOGEN (Pancreas) noncompetitive

Enteropeptidase

(intestinal cells)

TRYPSIN(+ hexapeptide)

(small intestines)

Prevents protein digestion in the pancreas.

(with pancreatic trypsin inhibitor)


Enzymes

The activity of digestive enzymes are typically limited noncompetitive

to the correct time and place by proenzyme activation.

1–6 +

7 ----- 245

1–6 – 7 ----- 245

1–13 – 14/15 – 16 --- 146 – 147/148 - 149 ----- 245

disulfide bonds hold 3 segments together


Types of enzyme regulation
TYPES OF ENZYME REGULATION noncompetitive

Reversible?

all or none?

extenal factor?

Yes

Yes

no

Allosteric

Covalent

Modification

Proenzyme

no

Yes

yes

allosteric regulator

protein kinase

protein phosphatase

activating protease