Alcohol metabolism – the role of KM

Cytosolic AcetaldehydedehydrogenaseKM value - high

Mitochondrial Acetaldehyde

reductaseKM value - low

CH3 CH2 OH NAD+

CH3 CHO

H+

NADHCH3 CHO

NAD+ H+

NADHCH3 COOH

+ + +

Alkoholdehidrogenáz

+ + +

Acetaldehiddehidrogenáz

TACHYCARDIA

Enzymes - KM values

Enzyme Substrate KM

Chymotrypsin Acetil-L-Trp 5000 mM

ß-Galaktosidase Lactose 4000 mM

Penicillinase Benzylpenicillin 50 mM

KM

1

32M k

kkK

E + S E + PES

k1

k2

k3

32 kk 1

2M k

kK

1

2ES k

k

[ES]

[S]*[E]K

KM ~ Dissociation constant for ES, if k2 >> k3

KM value is high: ES formation is weakKM value is low : assotiation

Importance of the Vmax value

Turnover number:

t3max [E]kV tmax3 /[E]Vk

Enzyme Turnover number / sec

Carbonic anhydrase 600000

Acetylcholin esterase 25000

Tryptophan synthetase 2

Inhibition of Enzymes

• Irreversible– Ser-OH– Cys-SH

• Reversible

CH2 OH

C

H

CH3H3C

O

P

O

OF

C CH3H3C

H

+

C

H

CH3H3C

O

P

O

O

C CH3H3C

H

OCH2Enzim Enzim

Diizopropil-fluorofoszfát

+ HFEnzyme Enzyme

Diisopropylfluorophosphate

Inhibitions of enzymes

Enzim EnzimCH2 SH ICH2 C NH2

O

+ CH2 S CH2 C NH2

O

HFMonojód acetamid

•-SH enzymes

+ HIEnzyme Enzyme

monoiodacetamide

Competitive inhibition of succinate-dehydrogenase by malonate

COO-

CH2

CH2

COO-

COO-

CH

CH

COO-

COO-

CH2

COO-

COO-

COO-

COO-

CH2

C

COO-

O

PO O

O

O

P

O

OO

FAD FADH2

Succinate-dehydrogenase

malonate

Oxalate

Oxaloacetate

pyrophosphate

Competitive inhibition

+ S

+ I

E + S ES E + P

I

EI

Ki

+

EI

I*EK i

Ki = dissociation constant for complex

Competitive inhibition of succinate-dehydrogenase by malonate

[Inhibitor 1]

[Enzyme]

[Inhibitor 2]

No inhibitor

Substrate+ competitive inhibitor

Competitive inhibition

+ S

+ I

E + S ES E + P

I

EI

Ki

+

EI

I*EK i

Ki = Dissociation constans of complex

+P

Competitive inhibition of succinate-dehydrogenase by malonate

Substrate [M]

Enzyme without ihhibitor

V [M/min]

Enzyme + inhibitor (1)

Enzyme + inhibitor (2)

Vmax

Vmax/2

Competitive inhibition

1/[S]

1/v

y=a*x+b

MaxMax

m

V

1

S

1*

V

K

v

1

MaxiMax

m

V

1

S

1*)]

K

I(1

V

K

v

1[

1/VMAX

Enzyme alone

E + inhibitor-1E + inhibitor-2

Competitive inhibition – Lineweawer-Burk plots

Kompetitív gátlás - Lineweaver - Burk ábrázolás

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35

1/[S]

1/v

Enzyme

Enzyme + Inhibitor 1

Enzyme + Inhibitor 2

Competitive inhibition

KM value - increase, Vmax - constant

KI =/= KS

Inhibition depends on [S]/[I].

Competitive inhibition

1/[S]

1/v

y=a*x+b

MaxMax

m

V

1

S

1*

V

K

v

1

MaxiMax

m

V

1

S

1*)]

K

I(1

V

K

v

1[

1/VMAX

Enzyme only

E + inhibitor1E + inhibitor2

CH3 CH2 OH CH3 C

O

H

CH3 OH H C

O

H

CH2 OH

CH2 OH

CH2 C

O

HCH2 COOH

ethanol acetaldehyde

NAD+ NADH + H+

alcohol-dehydrogenase

etanol formaldehyde

NAD+ NADH + H+

ethylenglycol glycolic acid

NAD+ NADH + H+

alcohol-dehydrogenase

alcohol-dehydrogenase

Methanol

Ethylenegly

col

Ethanol

Competitive inhibition

Acetylcholine esterase Acetylcholine Neostigmin Myasthenia gravisDOPA decarboxylase Dioxyphenyl-alanineMethyldopa HypertonyDihydrofolate reductaseDihidrofolate Aminopterine Leukemia

Xantine oxydase Xantine Allopurinol Gout

Noncompetitive inhibition

+ S+ I

E + I ES E + P

EI EIS

S

S

X

+ S

+ P

Noncompetitive inhibition

KM value is constant

VMax - decrease

Inhibitor-binding – different of substate binding site EIS complex – is inactive

Nonkompetitive inhibition

Kontroll

I2

I1

1/[S]

1/v

1/Vmax

-1/KM

Noncompetitive inhibition

E SH Cl Hg COO- Hg COO-SE+ HCl+

Metal / Enzyme + CN-, EDTA

Partially noncompetitive inhibitor

Both KM & Vmax values - decrease

1/[S]

1/v

Enzyme alone

I1

Uncompetitive inhibition

Inhibition of mixed typeInhibition of mixed type

KM - increase Vmax decrease

1/[S]

1/v

Control

I1

Turnover number

Number of molecules converted by 1 molecule of enzyme

Vmax = k3*[Et]

k3 [sec-1] i.e.: 10-6 M carbonic anhydrase 0.6 M of H2CO3 in 1 secundum

Enzyme Turnover number [sec-1]

Carbonic anhydrase 600.000,00

3-ketosteroid izomeráz 280.000,00

Acetylcholine esterase 25.000,00

Penicillinase 2.000,00

LDH 1.000,00

Chymotrypsin 100,00

DNA polimerase I 15,00

Tryptophane synthase 2,00

Turnover number

Activity units

Enzyme activity units: 1 E (U): 1 mole/min 25 oC 1 Catal (SI): 1 mole/sec

Specific activity: mo/min/mg protein mol/sec/mg protein

Enzyme activities – in everyday medical practice

Enzyme Measured value Normal value(l) Normal value (u).

Alk. phosphatase 175 U/l 98,00 290,00

CK 346 U/l 24,00 195,00

LDH 499 U/l 230,00 460,00

ASAT 21 U/l 1,00 46,00

ALAT 26 U/l 1,00 49,00

Akut szívinfarktust követő CK-MB és LDH aktivitás szérumban

0

100

200

300

400

500

600

700

800

900

0 24 48 72 96

Az infarktust követő idő [óra]

Enz

im a

ktiv

itás

[rel

atív

U]

CK-MB

LDH ö

Enzyme activities – in everyday medical practice