Biochemistry Basics

2007

BST

(1)
(2) Lehninger

Nelson & Cox (2005) Principles of Biochemistry

()

1 The foundation of biochemistry

1~3, 4

3 Amino acids, peptides, proteins

1 The foundation of biochemistry

2 Water

4 The 3-D structure of proteins

5 Protein function

8 Nucleotides & nucleic acids

2
3

9 DNA-based information technology

Introduction

Introduction

6 Enzymes

4
5

( 12 Biosignaling)

Kinetics
Inhibition
Mechanism
Regulation

7
8 9 DNA-based information technology

ii

2.1

2.2

2.3

3.1 pH

3.2

3.3

1
2

4
4.1

10

10

Ampholyte

pKa
4.2

10

12

()

14

15

1.1

15

iii

11

10

1.2

15

1.2.1

1.2.2
Turn
1.3

15

16

16

1.3.1

16

1.3.2

17

1.3.3
1.4

17

17

18

2.1

18

2.2

18

2.3

18

19

3.1

19

3.2

20

3.3.1
3.3.2 cDNA

19

3.3

20

20

3.3.3

20

21

23

Palindrome

RNA

25

Central Dogma

Intron exon

26

iv

PCR

DNA

RFLP

29

31

32

2.1

32

2.2

32

2.2.1

32

2.2.2

32

2.2.3

33

2.2.4 ribozyme

33

34

3.1
3.2

34
34

3.2.1

34

35

3.2.2 Michaelis-Menten

36

3.2.3 Michaelis-Menten

36

3.2.4 Vmax Km

37

3.2.4.1 Vmax Km

3.2.4.2 Km

37

3.2.4.3 Vmax

38

3.2.4.4
3.3

37

38

39

39

4.1
4.2

39
40

41

42

5.1

42

5.2

42

Carboxypeptidase A
5.3
5.4

43
44

42

Eadie-Hofstee

5.4.1

44

5.4.2
5.4.3

44

44

45

6.1

45

45

6.1.1
6.1.2

45

46

6.1.3 Ubiquitin-proteasome
6.2

46

47

6.3

47

6.3.1 cAMP 47
6.3.2 Calmodulin
6.3.3
6.4

47

47

48

6.4.1 Aspartate transcarbamoylase (ATCase)

S
6.4.2

7
7.1

49

49

50
50

7.1.1

50

7.1.2

50

7.1.3

50

7.2
7.2.1
7.2.2
7.2.3

51
51

51

51

7.2.4

52

7.3

52

53

8.1 (ELISA)

53

8.2

53

8.3

53

8.4 Proteome

54

8.4.1 Genome project

8.4.2 Proteome

54

55

vi

54

48

DNA

DNA
5'
process

ribosome
mRNA

Cell and Molecule

mRNA
mature
mRNA
3'
tail

cap
5'

proteins

5'

3'

proteins

(prokaryote)
(eukaryote)

1
a.



b.

c.

BCbasic 2007

molecular cell biology

2.1
(E. coli)
a. (cell wall) (peptidoglycan)
(1) (2) (3) ()
b. (flagella) (pili)
c. (cell membrane)
d. (cytoplasma) (ribosome)
e. (nuclear region) DNA
DNA () DNA (plasmid)
(vector)

2.2 (archaebacteria)

1) Methanogens
2) Halophiles
3) Thermacidophiles pH 2

2.3
(cellular organelles)
(nucleus)
a.
(nucleolus) RNA
(nucleoplasm) (chromatin) DNA
(chromosome)

b. (endoplasmic reticulum, ER)

RER (rough ER) SER (smooth ER)
RER (ribosome)

SER

BCbasics 2007

c. (Golgi body)
a.
b. (microbodies)
c. (glycoprotein)
d. (microbodies)
(1) Lysosome () (lysozyme)

(2) Peroxisome (catalase) H2O2

(3) Glyoxosome
e. (cytoskeleton elements)

f. (cell membrane)
(1) T
(2)
(3)
g. (mitochondria)
(1) (cristae)
ATP
(2) DNA

h. (chloroplast) (amyloplast)
(1)

(2) (plastid)
( proplastid)
(3) DNA

i.
(1) (cell coat)

(2) (microsome)
(3)

BCbasics 2007

Environmental pH

(amphoteric)
H+
(pH) ( 3)

11
10
9
8
7

Isoelectric point, 6
pI
5
4
3

Net Charge of Protein

3 pH

3.1 pH

a. H2O (
) ()
H2O H+ + OH-

H2O + H+ H3O+

b. H+ pH
pH pH

c. pH (buffering)
pH ( H+ )
H+ H+ pH
d. (dielectric constant)
(hydration)

3.2

a. (macromolecule)
(monomer)
b.

c.

(gene) (meme)

BCbasics 2007

3.3

(conformation) (affinity)
(secondary bonds)
a. (electrostatic bond)
b. (hydrogen bond)
()
c. (hydrophobic interaction)
-

d. (van der Waals force)

()

H
CNO

1s

N O

sp3

2s 2p

N O

sp

H2

CH4 NH3 H2O

C-C

O 3.5
N 3.0
C 2.5
H 2.1

- +
COOH

lp -

lp

NH 2
CH3

pH

4
BCbasics 2007

()

1. Stanley Miller

2.
3. helix
4.
5.
6. (eukaryotic cell) (prokaryotic cell)
7.
8.
9.
10.
11. Dawkins
(meme )
12. RNA
13.
14.
15. amphoteric
16.
17. A, T, C, G
18. ()
1)

(microbody)

2)

(microsome)

3)

4)

BCbasics 2007

Amino Acid

+
H3 N

COO

sp3

R group

L-form amino acid

1
a. ( carbon)
b. ()
c. (R) ( sp3 )
d. (D/L)
L D
e. R ( 1)
sp3
R
R

2
R
a. 1
R
b. R

c. R

BCbasics 2007

(1)

R=

Glycine

Gly G -H ()

P/N

Alanine

Ala

A -CH3

Valine

Val

V -C(C)-C

N*

Leucine

Leu L -C-C(C)-C

N*

Isoleucine

Ile

-C(C)-C-C

N*

Phenylalanine Phe

F -C-[C6H5]

N*

Tyrosine

Tyr

Y -C-[C6H4]-OH

Tryptophan

Trp W -C-[indole]

Histidine

His

Aspartic acid

Asp D -C-COOH

()

H -C-[imidazole]

(Asparagine) Asn N -C-CONH2

Glutamic acid Glu
(Glutamine)

N*
P*
P
P

E -C-C-COOH

Gln

Q -C-C-CONH2

K -C-C-C-C-NH2

P*

Lysine

Lys

Arginine

Arg R -C-C-C-[guanidine] P*

Serine

Ser

S -C-OH

Threonine

Thr

T -C(OH)-C

P*

OH-

Hydroxy Pro

Methionine

Met M -C-C-S-C

N*

Cysteine

Cys C -C-SH

(3) Cystine

(3)

Proline

-C-S-S-C-
Pro

P (imino acid)

Cys-Cys (2)
N

(1) * N, non-polar; P, polar

(2) (Cys) ()
(3)
2
R
Ala

BCbasics 2007

BASIC

R = C-C-C-N-C-N
N+

AROMATIC

Arg

R =C-

R = CN N+

OH

Tyr

Lys

R=
+
C-C-C-C-NH3

Trp

Polar

Non-polar

R =CN

R = C-CONH2

R = C-C-CONH2

Asn

Gln

AMIDE

Asp

Glu

ACIDIC

R =C-

His

Phe

Gly

Ala

R= H
R= C
OH
R = C-C
OH

R = C-COO-

Thr

Ile

Val
CC
R= C

R = CH3

Ser

R = C-C-COO-

Cys

C
R = C-C-C

R = C-SH

R = C-C-S-C

HYDROXYL SULFUR

C
R = C-C-C

Pro
Met

ALIPHATIC
Leu

R=

C
C C
HN C-COOH

IMINO, CIRCULAR

a. (peptide bond)
C-N C-N

b. (dipeptide)
(tripeptide) (polypeptide)
c. (Glu)

BCbasics 2007

4.1 (pKa)

(pKa) pKa 6~7

a.
( -COOH )
(-COO- H+) H+ ( -NH2)
(-NH3+)
b. Ampholyte
( -COO-) (
-NH3+) ampholyte ()
c.
pH pKa pKa
H+ 3
pKa pH pH pKa
(pH = pKa + log ([A-]/[HA]) 4

-COOH
R -COOH +
His -Imidazole-H
Cys-SH
Tyr -OH
+
-NH3
R -NH3+

-COO
R -COO
His -Imidazole
Cys-STyr -O -NH2
R -NH2

+
+
+
+
+
+
+

H
+
H
+
H
+
H
+
H
+
H
+
H

pKa
pKa
pKa
pKa
pKa
pKa
pKa

= 1.8-2.4
= 3.9-4.3
= 6.0
= 8.3
= 10
= 8.8-11
= 10-12.5

pKa

3 pKa

4.2 (pI)

a. pKa
pH pKa pI () ( pKa1 + pKa2)2 = pI
b. pH pI pH
Ampholytes
Zwitterion
10

BCbasics 2007

c. pH pH > pI
3 pH pI

d. R ( Lys ) pKa
H+ H+ pKa pKa pKa
pKa pI
pKa
e. pH
(AELKVGRRDV)
pH

AH A + H

- +
CH3COOH CH3COO + H

Ka
Ka =

+
[A- ] [H ]

[AH]

(pKa = 5)

10

Ka
log
+
[H ]
+
p -log (pH = -log[H ])

H-H

pH = pKa + log

[A- ]
[AH]

pH = pKa.... [A-] = [AH], log 1 = 0

pH

pH
pKa 5

[OH ]

[OH ]

4 pKa
Ka Ka
Henderson-Hasselbalch [A-][AH] pH pKa (= 5)

BCbasics 2007

11

1. peptide bond peptide

2.
3. Histidine pKa 1.8, 6.0 9.2 pI
4. Histidine pH
5.
Glutamate ()

Serine ()

Tryptophan ()

Cysteine ()

Proline ()
6.
Ser-Glu-Gly-His-Ala

Gly-His-Ala-Glu-Ser

7.
a.
b. R Ala, Val, Ile, Leu
c. R Asp, Lys
d. R Cys, Ser, Thr, Met
e.
f.
g.
h.
i.
j. His imidazole pKa 6.0
8. ()

______________ 2_______________

1_______________ 2_______________ 3______________

1_______________ 2_______________ 3______________
1_______________ 2_______________ 3______________
-COOH 1_______________

_______________

9. helix,
()
10. peptide Glu-Phe-Lys-His-Ile-Arg-Val
pH = 1 ____ pH = 7 _____ pH = 11 _____
11. ( R )
12

BCbasics 2007

12. ()
1) pH pH
Val His

Glu

Trp

2) pH 2 pH
Val His

Glu

Trp

3) (amphoteric)

DNA

4) pKa
pKa

pKa

pKa = 2

5)
CH4 glutamic acid

COOH

6)

13. 1_______________ 2_______________ 3______________
14. _____________
pH _______
15. pKa 5
16. DNA RNA

BCbasics 2007

13

14

BCbasics 2007

Protein

1.1

a. (primary structure)
N- (-NH2) C- (-COOH) H2N-C-C-[N-C-C]x-N-C-COOH
C N- C-

b.
DNA
c.
cDNA (cloning)
d. signal
peptide () signal
C- Lys-Asp-Glu-Leu (KDEL)

1.2

1.2.1
R

( helix) ( sheet) (secondary

structure)

BCbasics 2007

15

a. 3.6 Pro

(Ramachandran Plot)


b.
NC (parallel)
(antiparallel) R ( Ala, Gly, Ser)

c.
Pro ( PVPAPIPP) polyproline

1.2.2
a. Turn 180
turns turn ( Gly)
turn Pro Turns

b.
(random coil)

1.3
(globular)
1.3.1
a. (tertiary structure)

b.

c. Cys -SH (-S-S-)

1.3.2
a. , 88

16

BCbasics 2007

(motif)

b.
domain () domain
domains domain
domain
domain
c.
( RNase)
(native)
d. RNase
chaperonin
e.

1.3.3
a.
() (prosthetic group, heme)
(glycoprotein) (lipoprotein)
( )
b. (
)

1.4

a.
(quaternary structure)
(subunit)

b.
(positive cooperativity)

c.

BCbasics 2007

17

(
)

2.1
(denatuaration) pH

2.2
domain
domain

2.3

a. (conformational match) (
)
b. (interaction forces)
1

BINDING
SITE

a. Conformational Match:

Gly C=O...H-O Ser

(1)

Trp
Val
Leu (2) Ala
Asp

(3)

Lys

Contributed essentially by
van der Waals interactions

b. Interaction Forces:
(1) Hydrogen bond
(2) Hydrophobic interaction
(3) Electrostatic interaction
(4) Van der Waals interaction

18

BCbasics 2007

3.1

()
a.
(salting out)

b. (gel filtration)
c. (ion exchange)
pH

d. (affinity chromatography)

3.2
a. Coomassie Blue

Bradford method
b. (ultracentrifugation)
(gel electrophoresis) SDS

c. (pI) (isoelectric focusing)

pH

d. (HPLC)

e. X
(nmr)
f.
()

BCbasics 2007

19

3.3

3.3.1
2

Peptide

1
N-

PITC
(Edman degradation)

1-2-3-4-5-6---

2 Edman degradation
PITC (phenylisothiocyanate) N- PTH-
N- HPLC

a. ( Edman degradation) N-

b.

c.

3.3.2 cDNA
cDNA

3.3.3

20

BCbasics 2007

1. (hemoglobin) (myoglobin)

2.
3. DNA
Leu Val Lys Arg

4. (hydrophobic interaction)
5. (sickle cell)
6.
7. (urea)
8.
9.

10.
-LVRILNRILFFLWKTLTR11. domain
12.
a.
b.
c.
d.
e.
f. Sanger DNA Pauling double helix
g.
h.
i.
j. (irregular)
k.
l. Pro
m.

BCbasics 2007

21

n.

o.
p. Domain
q.
r.
s.
t. pI 5.2
13. ()
1) helix
helix
13 helix 13

Watson Crick
helix Pro

2)

R
3)

4)

DNA

14. 1____________ 2____________ 3___________

15.
16. Sanger, Anfinsen, Pauling, Edman, Kendrew & Perutz
17. helix
helix
18.

() (
)

22

BCbasics 2007


Nucleic Acids

DNA
RNA

1
a. (nucleotide)

()- 5' [] 1'-{} ( 1)

(nucleoside)

[] 1'-{}

(1) (ribose)
(deoxyribose) DNA RNA
(2) purine (A, G) pyrimidine (T, C, U)
T U
(3) ()

Acid

1
Monophosphate
Diphosphate
Triphosphate

Sugar
3

AMP, ADP dATP

b.

Base

Adenine
Purine
Guanine
Thymine
Cytosine Pyrimidine
Uracil

Ribose
Deoxyribose

Nucleo ide (Adenosine)

3'-OH 5'-

Nucleo ide (Adenosine monophosphate, AMP)

RNA
DNA DNA RNA
DNA A T G C Chargaff
c. (double helix)
DNA AT CG

(1) 5'3' 3'5'

(2) (large groove, L)
(small groove, s) 10.5

36 10.5
(3) pH DNA
DNA
(histone) DNA
(4) DNA A B Z DNA
BCbasics 2007

23

d.
DNA DNA
(supercoiling)
DNA DNA DNA
105 bp Voet et al. Fundamental
of Biochemistry (1999) p.733, Figure 23-9
(1) Fig. 23-9
DNA 10.5
twist (T) 105 bp DNA 10 (T)

L DNA linking linking number (L) DNA

twist linking () T = L 10 T 10 L
1 twist
10.5 bp

L = 10, T = 10 DNA W (writhing) = 0

(2) Fig. 23-9
DNA
(T = 9)
L = 9, T = 9 DNA W = 0
(3) Fig. 23-9
DNA T 9 T = 10 1 (-1) DNA
T (10) (W) L = T + W
(W = -1) DNA DNA
W ( L > T) positive supercoil
W (negative supercoil)

e. Palindrome
DNA GAATTC
EcoRI (restriction enzyme)
(cruciform) DNA DNA
DNA DNA (breathing)
DNA
f. (plasmid)
DNA DNA
DNA
(vector)
g. RNA
RNA RNA (mRNA) RNA (tRNA) RNA (rRNA)
24

BCbasics 2007

( translation) RNA
(ribozyme)
RNA RNA
DNA Central Dogma
h.
DNA mRNA template (-)
strand nontemplate (+) strand
DNA (promotor enhancer ) anti-sense RNA
(+) strand RNA mRNA
RNA (RNA interference, RNAi)

2 DNA
a.
(1) ATP ( GTP ) ATP
Glc-1-P UTP UDP-glc
(2) FAD, NAD+ coenzyme A (CoA)
(3) cAMP (second messenger)
b. Central Dogma
Central Dogma DNA RNA
DNA tRNA
mRNA Central Dogma
()
c.
DNA DNA 260 nm
hyperchromism
DNA (anneal)
nucleation ( DNA ) zippering (
)
d.
GC AT GC DNA (Tm)
DNA
DNA
DNA Cot
BCbasics 2007

25

e.
DNA DNA
(hybridization)DNA RNA
f. Intron exon
DNA DNA intron
mRNA exon RNA
intron (self splicing) RNA processing
()

3
a.

DNA DNA RNA

DNA 100 kb RNA
RNase RNase
b.
DNA

Amino acid sequence

GLY-ASP-GLU-SER-SER-VAL-LEU-----

()

GGG-GAC-GAG-TCC-TCC-GTT-CTC---

palindromeDNA

*
*
*
* *
* *
Nucleic acid sequence * Codon degeneracy

sticky ends ( cohesive

ends) (blunt

ends) sticky ends ligase ()

Synthesizing
oligonucleotide

PROBE: GGGGACGAGTCCTCCGTTCT

32

GGACG

AG

TC
CT
CC
GTTCT
A
CCCTGCTCAG
GC
C
T
GAGGCAAGAG
CA

c.

GG

DNA

DNA

GG

DNA
AT

DNA
denaturation

Target gene

Single colony

Southern blotting RNA

Northern blotting PCR

Lysed

DNA (
2)

d. (gene manipulation)
26

BCbasics 2007

DNA (ligase) (vector)

(molecular cloning)
DNA DNA

e. DNA
(1) cDNA
mRNA reverse transcriptase DNA
cDNA (complementary DNA) intron cDNA
cDNA
cDNA
(2) (genomic bank)
DNA
intron
( promotor, enhancer )
DNA
f. PCR (polymerase chain reaction)
DNA ( RNA) primers primers
DNA polymerase primers DNA

primers
g. DNA Sanger ( 3)
(1) Maxam-Gilbert

DNA
DNA
DNA
(2) Sanger
DNA DNA polymerase DNA
(analogue)
DNA
DNA
32P

BCbasics 2007

27

h. (site-directed mutagenesis)

i. RFLP (restriction fragment length polymorphism)

(polymorphism)
DNA DNA
DNA

Polyacrylamide Gel Electrophoresis

T
32

P
P
32
P
32
P
32
P
32
P
32
P
32
P
32
P
32

32

ATCGATCGAT
ATCGATCGA
ATCGATCG
ATCGATC
ATCGAT
ATCGA
ATCG
ATC
AT
A

G
C
T
A

3'-

Maxam-Gilbert's Method:
32

32

ATCGATCGAT

32

AT
ATCGATCG
ATCGAT
ATCG

Specific Reaction to G

Sanger's Method:
32

32

Template

ATCGA
TAGCTAGCTA

A,T,C,G Analogue

ATCG
TAGCTAGCTA

or

ATCG
TAGCTAGCTA

3
28

BCbasics 2007

1. DNA RNA
2. Human Genome Project
3. palindrome sequence
4.
5-AGGAGGATATACATGCAGAGTTAACTC-3
5. Bam HI GGATCC Bgl II AGATCT (
) ligase

6. ()
Adenine (), Adenosine (), Adenosine monophosphate (AMP),
Adenosine triphosphate (ATP), Deoxyadenosine ()
7. DNA
DNA
DNA
8. DNA RNA
a. DNA RNA
b. DNA
c. DNA
9. DNA
a.
b. DNA
c. DNA ( supercoiling)
10. DNA
DNA
11. genomic bank cDNA bank

12. Central Dogma DNA RNA Protein

(macromolecule)
a.
b. ()
BCbasics 2007

29

13. DNA A=T

CG DNA
(denatured)
a.
b. DNA hyperchromic effect
c.
14. DNA RNA
(2'-OH)

15. DNA (transcription)

16. AUG Met

Met Met

17.
(vector)
18. intron exon intron intron exon

19. DNA RNA

DNA superciol
20. DNA G C DNA
21. RNA -AAUAGGUACC- RNA
sense DNA
22. RNA
23. DNA RNA
(a) (b)

(c) pI (d)

24. RNA ribozyme RNA

25. (genome)

30

BCbasics 2007

Enzyme

Enzyme in yeast
Sumner 1926 (urease)

a.
b.

c.
d. RNA (ribozyme)

a. -in -zyme trypsin, renin

lysozyme -ase
histidine decarboxylase ( + -ase)
b. 1965 (IUBMB
) histidine carboxylase EC 4.1.1.22
Main Class
Subclass
Sub-subclass

4
4.1
4.1.1
22

Lyases
C-C lyase
Carboxylase
22 4.1.1

C-C, C-O, C-N

C-C
C-COO
C-COO

c. IUBMB Main Classes

(1) Oxidoreductase

(2) Transferase

(3) Hydrolase

(4) Lyase

(5) Isomerase

(6) Ligase

ATP

BCbasics 2007

31

2
RNA

2.1

a.
(cofactor, coenzyme) ( holoenzyme)
apoenzyme
Holoenzyme Apoenzyme Cofactor/Coenzyme
b. (
chymotrypsin) (subunit)
(dimer) (hemoglobin) 22
(allosteric effect)

2.2

2.2.1
()
a. Zn2+, Mg2+, Mn2+, Fe2+, Cu2+, K+ His, Cys, Glu

b. (coenzyme)
B (folic
acid) (niacin)
2.2.2

a.
b.
( -CH3, -CO2, -NH2)

32

BCbasics 2007

c. B1 (thiamine)

2.2.3

a. (dehydrogenase) NAD+/NADH (hydride, H-)
alcohol dehydrogenase glyceraldehyde-3-P dehydrogenase
NAD+/NADH
b. Carboxypeptidase (induced fit)

c. Glutamate transaminase pyridoxal phosphate

d. Catalase Fe2+ H2O2
Fe2+
Fe3+
2.2.4 ribozyme
a. RNA
RNA RNA
ribozyme

b. ribozyme
RNA

+-

BCbasics 2007

33

3.1

a. (A, B) (A-B) [A...B]

A + B [A...B] A-B
b. (transition state) (activation
energy, Eact)

c. (analog)

abzyme (catalytic antibody)

d.
(1)
(2)
(3) ()
(4) ()
1
( Gonick, L.
& Wheelis, M. The Cartoon Guide to Genetics, )

3.2

3.2.1
1913
Michaelis Menten (invertase)

a. Steady state
(E + S
ES)
(steady state) [ES] ( ES )
b. (v)
34

BCbasics 2007

v [S] ()
Michaelis-Menten (M-M)

Juang

3.1

3.2

E+S

k1
k2

ES

k3

E+P

Steady State [ES] 3.2.1

ES

k1 [E][S] = k2 [ES] +k3 [ES]

Michaelis-Menten

[Et] = [Ef] + [ES]

vo = k3 [ES]
Vmax = k3 [Et]

3.2.2
3.2.3

kcat Km

vo =

Vmax [S]
Km + [S]

kcat
Turn over
number

3.2.4

zero order

3.2.4.1

1st order

[S]
vo

Vmax Km

E3
E2
E1

3.2.4.3

k3 [Et]
Activity Unit
1 mole
min

unit
mg

Vmax

&

Km
4

3.2.4.4

Competitive

3.2.4.2

3.3

M-M

Non-competitive

Uncompetitive

1
Gonick, L. & Wheelis, M. The Cartoon Guide to Genetics.

BCbasics 2007

35

3.2.2 Michaelis-Menten
M-M
a. E S k1, k2, k3
k1
k3
E+S
ES E + P
k2
(vo)
b. M-M
(1) [ES] ES
k2 [ES] + k3 [ES] = k1 [E][S]

(I)

(2) [Et] [Ef] + [ES]

(II)

(3) (vo) (k3) vo = k3 [ES]

(III)

(4) (Vmax) [ES]

Vmax = k3 [Et]

(IV)

c. M-M
(1) (I) (k2k3) [ES] = k1 [E][S]

[ES]

k1
k2k3
[E][S]
[ES] = [E][S] Km [ES] =
k2k3
k1
Km
vo
vo
[E][S]
k3 [E][S]
(2) (III) [ES] = vo
k3
k3
Km
Km

Km
[ES]
vo

(V)

(3) (II) [Ef] [Et] - [ES] [Ef] [E]

[E]

[E] [Et] - [ES] (V)

k3 [Et][S] - k3 [ES][S]
k3 ([Et]-[ES])[S]
vo
Km
Km
(4) (III) vo k3 [ES] (IV) Vmax k3 [Et]

Vmax vo

Vmax [S] - vo [S]

vo vo Km Vmax [S] - vo [S]
Km
vo Km + vo [S] Vmax [S]

(VI)
vo

(VI) vo M-M

Vmax [S]
vo
Km + [S]

3.2.3 Michaelis-Menten

vo

M-M Vmax Km Vmax Km

a. M-M [S]

36

[S]
BCbasics 2007

vo [S] x vo y

vo

zero order

Vmax
b. [S] vo [S] vo[S] 1

1st order

[S]

(first order reaction) [S] vo vo

[S] vo [S] 0 (zero
order)
c. [S] M-M

vo

E3
E2
E1

Vmax ( [S])
vo Vmax ()
( Km) + ( [S])
(IV) Vmax = k3 [Et] vo[Et]

[S]

d. ES E + P M-M
(vo) [P]

M-M
3.2.4 Vmax Km
Vmax Km
3.2.4.1 Vmax Km

[S] (vo)
Vmax Km
a. [S] vo
VmaxKm 50% Vmax [S]
1/vo

b. Lineweaver-Burk
Vmax x y 1/[S] 1/vo
x 1/Km y 1/Vmax

1/[S]

Vmax Km
c. Eadie-Hofstee y
vo/[S] vo x, y

3.2.4.2 Km
Km Km
a. 50% Vmax vo = Vmax M-M

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37

Vmax
Vmax [S]
= Km [S] ( vo Vmax )
2
Km[S]
Km [S]
b. Km Vmax
Km Km
Km [S] (mM M)
c. Km
3.2.4.3 Vmax
Vmax = k3 [Et] ( IV)
a.
Vmax Vmax
Vmax
b. () turn over number
molecular activity 0.110,000 ()
Km ([S] >> Km)E + S ES E
+ P k3 turn over number kcat
c. Km (Km >> [S] [Et] = [E] Km + [S] = Km)
M-M
Vmax [S]
k3 [Et][S]
k3 [E][S]
kcat
vo [E][S]
Km + [S]
Km + [S]
Km
Km
second order [E] [S]
kcat/Km
d. Km Vmax
k1
k3
E+S
ES E + P
k2
(vo)
E + SES k1 k2 ESE + P
k3 Vmax ( Vmax = k3 [Et]) Km

Km
Km (k2+k3)k1 Km
3.2.4.4

a. (unit)
1 mole 1

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BCbasics 2007

b. (mg) (unit)
(specific activity, unit/mg)

3.3
a. SP Uni-Uni

S1 S2 P

(Bi-Uni, -)

(Uni-Bi, -)

P1 P2

S1 S2 P1 P2

(Bi-Bi, -)

b. M-M Km S1
S2 ( S2 )
c. Bi-Bi (S1, S2) (P1, P2)
(1) Random

(2) Ordered sequential

(3) Ordered ping-pong [S1 , P1 ; S2 , P2 ] Uni-Uni

BCbasics 2007

39

4.1
a.
b.
(PABA)

c.

d. [I] [E]
Competitive, non-competitive uncompetitive ( 2 )

4.2

a. (penicillin)

b. Hg2+, Pb2+, Cd2+ [E] [ES]

c.
( 1)
1

PCMB

p-chloro-mercuribenzoate

Cys -SH

Papain

DIFP

diisopropyl-fluorophosphat

Ser -OH

Ser proteases

Ser -OH

chymotrypsin

Ser -OH

Trypsin

TPCK
TLCK

tosyl-L-phenylalanine
chloromethyl ketone
tosyl-L-lysine chloromethyl ketone

(Sarin) DIFP

40

BCbasics 2007

d.
HIV

6.1.2

Competitive

Non-competitive

Uncompetitive

E+S
+
I

X
I

E + S ES
+
+
I
I

E+P

EI

EI + S IES

[I] [E]
[S]
[S] [I]

[I] [E]
[S] [ES]
[S] [I]

Vmax

Vmax

vo

Km

[S], mM

[S], mM

1/vo

1/vo

1/Km

1/[S]

[I] [ES]
[S] [I]

Vmax

vo

Vmax'

Km' Km

[S], mM

Vmax Km
1/vo

1/Vmax

E+P

Vmax Km

E + S ES
+
I

Vmax'

Vmax Km

IES

Km Km'

ES E + P

vo

1/Vmax
1/Km

1/Vmax
1/[S]

1/Km

1/[S]

BCbasics 2007

41

5.1 (active site)

a. ()

(reactive group)
b.
(1) Bond strain

(2) Acid-base ()

(3) Orientation
(concerted set)
(sequential mechanism)

5.2
carboxypeptidase A (CPA, ) ( 3)
a. CPA 34 kD 307
CPA C- () R
carboxypeptidase B (CPB) C- Lys Arg

Carboxypeptidase A

(248)
Tyr

(270)
Glu 3

COO +

H
H O-

C
2
1

His
(196)

OH

ACTIVE
SITE

O-

+
Zn

His (69)

COO Glu
(72)

+Arg

(145)

C-terminus

C-

3 Carboxypeptidase A
42

BCbasics 2007

b. CPA 3
Zn2+ carbonyl

Glu270 OH- C+ C-OH

Tyr248-OH lone pair
C- R
Arg145 C- -COOH C-

5.3
chymotrypsin (CT, )
a. CT 25 kD
N- Arg15 Ile16
CT
b. CT (Tyr, Phe, Trp) Met (
) C-
c. CT
(Ser195, His57, Asp102) Ser
Ser195-OH H+ His57 O -
Asp-CO-O -. H+. N His
102
57

N.H+. -O -Ser
195

4 Ser
d. Ser O- carbonyl ()
CO (acylation ) C-
CO (deacylation ) N-

e. catalytic triad
Gly193 Ser195
N-H
f. Ser Ser serine
chymotrypsin trypsin ()elastase ()
[Ser-His-Asp]
trypsin (Lys, Arg)elastase R

BCbasics 2007

43

5.4

5.4.1
CT

a. Ser [Ser-His-Asp]
()
b. R
chymotrypsin

(Science, 1992, 255: 1249)

Van der Waal interactions

5.4.2

a. lock & key

vdw

vdw

induced fit
5 A B

b.

between non-polar surfaces

5.4.3

a. ( L
D )

b. 6 (sp3) (A)
B-C-D
( B-D-C) ( A

sp3

7)

C
B

(-)
D

(+

44

BCbasics 2007

(6.1~6.4)

6.1
or

proteolysis

inhibitor

6.4

6.2
R

o
S

o
S

(+)

regulator
effector

phosphorylation

6.3

x
(-)

A or

o
S

cAMP or
calmodulin

8 ()

6.1

6.1.1

a. () mRNA
(zymogen) (precursor)

*
(1) Prothrombin Thrombin* []

cascade
(thrombin)

(2) Preproinsulin Proinsulin Insulin*

preproinsulin N-
BCbasics 2007

45

proinsulin C
(3) trypsin

b. ()
(compartmentation)
Chymotrypsinogen -Chymotrypsin* -Chymotrypsin*
Chymotrypsinogen N- Ile16
-NH3+ Asp194 Ser195

c.
(inhibitor) Trypsin trypsin
()
6.1.2

a. (1) (2) Serine (3) Cysteine ( thio)

(4) Aspartyl ( acid)
b.
( Ser catalytic triad)
acetylcholinesterase
catalytic triad ester
c. intron exon intein extein

6.1.3 Ubiquitin-Proteasome

ubiquitin proteasome
a. Ubiquitin ()
()
Lys (ubiquitination)
N- (destruction box)
b. Proteasome ()

ubiquitin ubiquitin

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BCbasics 2007

6.2 (phosphorylation)

(glycogen
phosphorylase, GP)
a. Ser, Thr, Tyr ( His) -OH (His imidazole )
(protein kinase)
(protein phosphatase)
b.

c. ubiquitin-proteasome

6.3
cAMP calmodulin

6.3.1 cAMP
cAMP cAMP

(, catalytic subunit; , regulatory subunit; = + cAMP)

+ 4 cAMP
[]

+ *

+ 4 cAMP

6.3.2 Calmodulin ()
calmodulin
(17 kD)

6.3.3

cAMP

BCbasics 2007

47

Signal Receptor Transducer Effector enzyme Effector Effect

Glucagon Receptor G-protein Adenylate cyclase cAMP Kinase

a. (amplification)
( cyclase, kiase)

cascade
b. (flexibility)

6.4 (allosteric enzyme)

(feedback)

(regulatory site)
6.4.1 Aspartate transcarbamoylase (ATCase)
ATCase
a. ATCase aspartate carbamoyl-P carbamoyl aspartate
CTP CTP ATCase
ATCase () ATCase ATCase

b. ATCase (catalytic subunit, CCC)

(regulatory subunits, RR) (C, 34
kD) (R, 17 kD)
2(CCC) 3(RR) C6R6

CCC
R R R
R R R
CCC

c. S ATCase vo [Asp]
S (sigmoidal curve) M-M ( 9)
(cooperative)
(positive homotropic
effect)

48

BCbasics 2007

d. S

Vo

Noncooperative
(Hyperbolic)
ATP

CTP

Cooperative
(Sigmoid)

Vmax ( 9
)
e. CTP 9 S

Vo

( Vmax ) CTP ATCase

(negative heterotropic) ATP

ATCase (positive heterotropic) S

M-M
f. ATP CTP
(effector)
Asp
(

Off
On
[Substrare]

)
ATP CTP
6.4.2

a. () T (tense)
R (relaxed)

(1) Concerted T R
RR TT RT
TT RR

(2) Sequential ()
T R ( T
)
TTRTRR
b.
R
() homotropic ()

() heterotropic ()

BCbasics 2007

49

7.1

7.1.1

a.

b.
(rate-limiting step)

c.
ATP
d.

7.1.2

(glycolysis)
a. Stage 1: () (
)
b. Stage 2: acetyl CoA
c. Stage 3: Acetyl CoA CO2
7.1.3

a. pyruvate acetyl CoA ()

b. acetyl CoA CO2 NADH
c. NADH ATP

50

BCbasics 2007

7.2

7.2.1

a. (DNAmRNAprotein)

(gene expression) mRNA

mRNA
b. induction
repression (operon)
repressor lac operon
7.2.2

a.
cAMP

b.
cascade ()
cascade (amplify)

ubiquitin proteasome
7.2.3

a. ( insulin)
(receptor)

()
b.

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51

7.2.4

a. (compartmentation)

b.

pyruvate dehydrogenase ( pyruvate acetyl CoA)

c.

ATP

7.3

a. (homogeneous enzyme)

b.
(cell-free lysate)
(callus)
(organelle)
c. (whole organism) (organ)

d.

e.

52

BCbasics 2007

8.1 (ELISA)

(solid
phase) ELISA (enzyme-linked immunosorbent assay)

()

- 1 ()...[]... 2 ()...[ ()-]

8.2

1a

1b

2 E
2nd Ab

a.

10 ELISA

(1)
(2) pH
(3)
(4)
b.
( pH )

8.3

a. molecular cloning
cDNA cDNA (
) DNA
( luciferase, GFP GUS)
(fusion protein) reporter

BCbasics 2007

53

b.

(site-directed
mutagenesis)
c.

d. Abzyme ()

abzyme
(Nature 1996, 383: 23-24)

8.4 Proteome

8.4.1 Genome project

DNA
(Human Genome Project) DNA J.D.
Watson

8.4.2 Proteomics
a. DNA

(proteome)
b.
(bioinformatics)
c.

(metabolomics)
d.

(systems biology)

54

BCbasics 2007

1. Hemoglobin H2O2
2. RNA
3. Carboxypeptidase A Zn
4. ATP, Coenzyme A, NADPH, Thiamine
5.
6. Glyceraldeyhyde-3-P dehydrogenase domains
7.
8.

9. Michaelis-Menten
10. k3 turnover number
11. kcat/km
12.
a) Penicillin b) Sarin c) Sulfa drug

d) Heavy metal

13. chymotrypsinogen
14. Chymotrypsin
15. Chymotrypsin His57 His57 pH
16. DPF (diisopropyl phosphofluoridate) Ser protease
DPF
17. proteases
18. proteases protease

19.
Chymotrypsinogen
20.
21. (allosteric enzyme) S S

22. cascade
23. pH pH
BCbasics 2007

55

24.

25. catalytic
antibody ( abzyme)
AB A-B (A B )
a) (hint: )
b)
26. ()
1)
2)
3) Vmax
4)
5) Km /
6) (inhibitor) (negative effector)

7) ( Km )
8) k3 = kcat = turnover number
9) Km [ES]
-

10) Ser proteases Asp-His-Ser triad -O

11)
12)
13)
14)
15) AMP
16) (protein kinase)

17)
18)
19)
20)

21)

56

BCbasics 2007

22) S
23) (hemoglobin)

24)

25) chymotrypsin
26)
27) (urea)
28) mRNA

29) ---
30) RNA DNA DNA
31) RNA DNA RNA Watson-Crick
32) k3 (vo = k3 [ES]) k1 k2
33) Steady state
34) serine protease
35)
36)
37)

38) abzyme
39) domain
40) Carboxypeptidase A C-
-COOH C-
27. ()
1)

2) RNA
RNA
pairing

RNA Watson-Crick pairing RNA W-C

RNA 2'-OH

3)
Insulin Thrombin Fibrin
BCbasics 2007

Hemoglobin Calmodulin
57

4)
Val His

Glu

Pro

5) NADH

Adenine Hydride

Nicotinamide

6)

7) Km
Km mM/sec

Km = k2(k1 + k3) Km

Km Km ES
8) kcat
kcat sec-1

kcat k3

kcat

kcat ES

9)

10) Ser protease

Subtilisin VIII Acetylcholinesterase Elastase Thrombin
11)
Penicillin PCMB DFP EDTA

cAMP

12) chymotrypsin
Asp102

His57 Ser14 Ile16 C-terminal

13)
sp3 L-

14)

cAMP (+) ATP

15)

effector

28. E + S ES E + P

29. trypsin
58

BCbasics 2007

30.
31.
32.
a)
a

b) a b
c) X

33.
a) _____________________
b) _____________________
c) _____________________
34. Abzyme, Metabolomics, Ribozyme, Proteasome, Specific activity
35.
36.
37.

38. amplification
(cyclase, kinase)
39.
40. (
)
()

BCbasics 2007

59