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1 protein &
enzyme
structure & function
(1) amino acid & peptide
1) amino acid
1
A、Biomedical
importance
• 10 essential amino acids
• perform a multitude of
structure,hormone,catalitic
functions essential for life.
2
• several comparatively rare
genetic disease of amino
acid catabolism
•
amino acid urines
• some intracellular functions
3
B 、properties of amino acid
• the genetic code specifies 20
L--amino acids
• additional amino acid occur
in specific proteins
4
• only L--amino acids occur
in protein
COOH
NH2
C
R
H
5
6
• mammals contain certain
free D-amino acid
• amino acid may have positive,
negative, or zero net charge
7
(1) zwitterions
NH 3+
O-
R
O
8
(2) isoelectric pH (pI)
H+
R-CH-COO-
H+
R-CH-COO-
0H
NH2
R-CH-COOH
0H
NH3+
NH3+
9
• the classification of amino
acid by R group
(1) hydrophobic & hydrophilic
(2) acidic & basic
(3) aromatic & aliphatic
10
11
• the  -R group determine the
properties of individual
amino acid
glycine, aliphatic R group, charged R
group, alcohol group, thioalcohol group
12
• The ultraviolet absorption
13
14
• various techniques
separate amino acids
Chromatography
electropheresis
15
16
17
• functional groups dictate the
chemical reactions of amino acid
the most important reaction of
amino acids is peptide bond
formation
18
2) peptides
A biomedical importance
• Hormone
• Nervous system
• antibiotics
19
B L--amino acids linked by
peptide bonds form
peptide
• peptide bond
20
The formation of peptide
bond
the peptide bond has partial
double-bond character
21
22
23
24
25
• peptide are chain of amino
acid
• peptide can contain
unusual amino acid
26
27
28
29
(2) The structure of protein
primary structure
three dimensional structure
30
• the three dimensional
structure of a protein is
unique
31
• the function of a protein
depends upon its three
dimensional structure
32
•
three dimensional
structure means
conformation
33
• Configuration
& conformation
34
1) The primary structure
• definition
• linkage bond
• importance
• example
35
36
2) The secondary structure of
protein
• definition
•
types
37
a.
 -helix structure
• the basic characteristics
38
39
40
41
b. -helix
• the basic characteristics
42
43
b.  –pleated sheet
structure
• the basic characteristics
44
45
46
c.  -turn
• the basic characteristics
47
48
d. Random coin
• the basic characteristics
49
3) Two addition structural
levels intermediate
between secondary &
tertiary structure
• Super secondary structure
• domain
50
• the basic characteristics
• importance
51
52
53
54
55
4) the tertiary structure of
protein
• definition
• the basic characteristics
56
57
• importance
58
59
5) the quaternary structure
of protein
• definition
• the basic characteristics
 subunit
60
61
62
6) Bonds responsible for
higher levels of protein
structure
• covalent bond
• noncovalent bond
63
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(3) The relationship
between structure &
functions
1) the primary structure &
function
65
• the different primary
structure ,the different function
• the similar primary
structure , the similar function
66
• the alteration of primary
structure of the proteins
causes molecular disease
• sickle cell anemia
67
2) The relationship between
three dimensional structure
& juncture
• the special conformation,the
special function
68
• allosteric effect
* definition
* significance
* instance
69
70
71
(4) the classification of
protein
 simple protein
* conjugated protein
* fibrous protein
* globular protein
72
(5) The physic-chemical
properties of protein
• Macromolecular & colloid
properties
polyvalen zwitterions
(amphopathic molecules)
73
• UV absorbance &
quantitative analysis
• precipitation
 conception
74
 mechanism
 methods

salting out

organic

heavy metal
reagent
75
• denaturation
 conception
 factors
 mechanism
 change of physicalchemical properties
76
 characteristics
77
78
 application
 the differences
between allosteric
effect & denaturation
79
Enzyme
Structure & function
80
1. Introduction of
enzyme
1853
Tour, Schwann
1878
Kuhne
1847
E.Bucher
1926
Summer
1982
Cech
81
2. The concept of enzyme
82
3. The differences between
biologic catalyst &
inorganic catalyst
83
• high efficiency of
catalysis
• high specificity
• high instability
84
2H2O2
2+
Fe
2H2O+O2
6
mol/mol.S
-4
10
6
10
peroxidase 5
mol/mol.S
6
12
10 -10 倍
85
4.The structure of
enzyme & catalytic
activity
• the chemical composition
of enzyme
86
• simple enzyme
• holoenzyme
• apoenzyme
• cofactor
87
 metal ions
 family of vitamin B

coenzyme

Prosthetic group
88
5. The chemical structure &
catalytic function
• the active site of the enzyme
essential group
 Binding group


Catalytic group
89
90
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• zymogene & activation of
zymogene


conception
method of activation
92
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 the hypostasis &
significance
• multienzyme complex &
multifunctional enzyme
94
95
• isoenzyme
 conception
 the research of isoenzyme
 the damnification of
specific tissue
96
lactic dehydrogenase,LDH
a.catalytic reaction
b.The molecular
structure
97
MW, the type & the
number of subunits,
The physical-chemical
properties
98
99
 estimate the pathologic
changes of the tissue
• apart from the active site
the other structure
concern the catalytic
activity
100
 allosteric enzyme
a.concept
b.allosteric activator
allosteric inhibitor
101
c. the characteristics of the
molecular structure
a) oligomers
b) binding site,catalytic site
102
c) the position of the
metabolic pathway
103
Allosteric enzyme
substrate
intermediate
product
feedback
104
d) the curve of
substrate concentration
105
106
e) Biologic significant
 Modification enzyme
a) concept
b) instance
107
glucogen
(Gn)
glucogen + glucose
(Gn-1)
phosphorylase
Active(+P)
Inactive(-p)
108
6. Enzyme classification &
nomenclature
7.the mechanism of enzyme
action
109
• Formation of an enzymesubstrate complex is the
first step in enzymatic
catalysis
110
 Binding energy
 transition state
111
112
E+S
ES
ES’
E+P
unstable
• decrease the activation
energies of reaction
113
 active molecule
 active energies
114
115
116
• the theory of the the
transition state
• Special catalytic group
contribute to catalysis
117
118
9.the kinetics of enzymecatalyzed reaction
119
• The facts affected
the rate of
enzyme-catalyzed reaction
substrate
pH
enzyme
activator
temperature
inhibitor
120
• how to mensurate the
rate of the enzymecatalyzed reaction
121
• the precondition of the
research the enzymecatalyzed reaction
The initial velocity of
the reaction
122
1) Enzyme concentration
affects the rate of enzymecatalyzed reaction
v=k[E]
v
[E]
123
2) substrate concentration
affects the rate of enzymecatalyzed reaction
124
• 1913
Michaelis Menten
125
[S ]lower
[S]
vo
vo=-d[S]/dt =K[S] First
order
[S] higher
[S]
vo
vo=-d[S]/dt =K[S] First
order
[S]
saturated
[S] vo
vo=-d[S]/dt =K
unchanged
Zero
order
• rectangular hyperbolic plot
126
127
• Michaelis-Menten
equation:
K1
E + S <---> ES
K3
EP
E+P
K2
• Michaelis-Menten constant (Km)
128
vo = Vmax[S] /Km +[S]
[S] << Km, vo = Vmax[S] /Km
[S] >>Km
vo = Vmax[S] / [S]
[S] =Km
vo = Vmax[S] / 2[S]
= 1/2Vmax
129
• the definition of Km
• the significance of Km
130
Reflection of the
affinity between
substrate & enzyme
 Calculate the concentration of the
substrate (which can reach the how
many percentage of Vmax) or
velocity of the reaction ( at the
certain [S] )
131
K1
E+S
K3
ES
E + P
K2
( K2 + K3 ) / K1 = Km
If
K2 >>K3
Km = K2 / K1 = [ E ] [ S ] / [ ES]
here , Km defined as dissociation constant
132
• the mensuration of the
Km & Vmax
 double reciprocal plot
(lineweaver-burk plot)
133
V0= Vmax [S] / ( Km + [S] )
Km 1
1

1/v0 =
+
Vmax [S]
Vmax
134
135
3) enzyme activity is affected
by temperature
• optimum temperature
136
v
T
optimum temperature
137
138
4) enzyme activity is affected
by pH
• optimum pH
139
140
5) enzyme activity is
affected by activators
• essential activators
• nonessential activators
141
6)
enzyme activity is
affected by inhibitors
• irreversible inhibition
 specific
142
143
acetylcholine estarase
Acetylcholine
acetic acid + choline
144
145
 nonspecific
146
 Leurisle gas & anti
leurisle gas
147
SH
Cl
E
+
SH
AS-CH=CHCl
Cl
S
E
AS-CH=CHCl + 2HCl
S
+
OH-CH2-CH-SH
CH2-SH
SH
E
+
SH
OH-CH2-CH-S
AS-CH=CHCL
CH2-S
148
• reversible inhibition
 Competitive
inhibition
149
150
 characteristics
K1
E+S
+ K2
I
K3
ES
E+P
EI
Vo
Km
Vm
 instance
unchanged
151
152
 double reciprocal plot
153
 Clinical application
154
angiotensin convert enzyme (ACE)
angiotensionⅠ
angiotensinⅡ
155
 noncompetitive inhibition
 characteristics
156
157
K1
E+S
+ K2
I
EI+S
V
, Km
K3
ES
+
I
E+P
ESI
unchanged , Vm
158
159
 uncompetitive inhibition
 characteristics
160
161
K1
E+S
K2
K3
ES
+
I
E+P
ESI
V
, Km
,
Vm
162
163
Inhibit
or Type
Binding Site on Enzyme
Kinetic effect
Competiti
ve
Inhibitor
Specifically at the catalytic site, where it competes with
substrate for binding in a dynamic equilibrium- like process.
Inhibition is reversible by substrate.
Vmax is unchanged; Km, as defined by [S]
required for 1/2 maximal activity, is
increased.
Noncompe
titive
Inhibitor
Binds E or ES complex other than at the catalytic site.
Substrate binding unaltered, but ESI complex cannot form
products. Inhibition cannot be reversed by substrate.
Km appears unaltered; Vmax is decreased
proportionately to inhibitor concentration.
Uncompet
itive
Inhibitor
Binds only to ES complexes at locations other than the
catalytic site. Substrate binding modifies enzyme structure,
making inhibitor- binding site available. Inhibition cannot be
reversed by substrate.
Apparent Vmax decreased; Km, as defined by
[S] required for 1/2 maximal activity, is
decreased.
164
 characteristics
165
10 Enzyme activity detecting
& enzymatic unit
 In disease diagnosis
 In disease treatments
166
Serum Enzyme
Major Diagnostic Use
Aminotransferases
(AST)
Myocardial infarction
(ALT)
Viral hepatitis
Amylase
Acute pancreatitis
Ceruloplasmin
Hepatolenticular
degeneration (Wilson disease )
γ-glutamyl transpeptidase (γ-GT)
LDH ,CK
lipase
Phosphatase ,acid
Phosphatase, alkaline
various liver disease
myocardial infarction
Acute pancreatitis
Metastatic carcinoma of the prostate
various bone disorders,
obstructive liver disease
167
1. 组织受损或细胞膜通透性增加
Acute pancreatitis（amylase），
Myocardial infarction（LDH1，CPK2）
Viral hepatitis （ALT，LDH5）
2.体内代谢障碍
rachitis （AKP）
3.酶排泄障碍
obstructive of bile duct （AKP）
168
4. 酶合成障碍
liver disease （prothrombin）
5.肿瘤
Metastatic carcinoma of the prostate
（ Phosphatase ,acid，ACP ）
liver cancer（ γ-glutamyl transpeptidase，γ-GT ）
metastasis of cancer （LDH）
169
6.酶活性下降
toxicosis of pesticide
（ acetylcholine estarase ）
cyanide
（cytochrome oxidase）
170
治疗应用：
助 消化 (多酶片）
消炎（胰，糜蛋白酶）
抗凝 （链激酶，尿激酶）
抗肿瘤 （天冬酰胺酶）
其它
（超氧歧化酶，右旋糖苷酶等）
药物酶的问题
171