simple protein * conjugated protein* fibrous protein
* globular protein
73
(5) The physic-chemical
properties of protein
• Macromolecular & colloid
properties
polyvalen zwitterions
(amphopathic molecules)
74
• UV absorbance &
quantitative analysis
• precipitation
conception
75
mechanism
methods salting out
organic reagent
heavy metal
76
• denaturation
conception
factors
mechanism change of physical-
chemical properties
77
characteristics
78
79
application
the differences
between allosteric
effect & denaturation
80
Enzyme
Structure & function
81
1. Introduction of
enzyme
1878 Kuhne
1847 E.Bucher
1926 Summer
1982 Cech
1853 Tour, Schwann
82
2. The concept of enzyme
83
3. The differences between
biologic catalyst &
inorganic catalyst
84
• high efficiency of
catalysis
• high specificity
• high instability
85
2H2O2 2H2O+O2
Fe2+ 6 10-4
mol/mol.S
peroxidase 5 106 mol/mol.S
106-1012 倍
86
4.The structure of
enzyme & catalytic
activity
• the chemical composition
of enzyme
87
• simple enzyme
• holoenzyme
• apoenzyme
• cofactor
88
metal ions
family of vitamin B
coenzyme
Prosthetic group
89
5. The chemical structure &
catalytic function
• the active site of the enzyme
essential group
Binding group
Catalytic group
90
91
92
• zymogene & activation of
zymogene
conception
method of activation
93
94
the hypostasis &
significance
• multienzyme complex &
multifunctional enzyme
95
96
• isoenzyme
conception
the research of isoenzyme
the damnification of
specific tissue
97
lactic dehydrogenase,LDH
a.catalytic reaction
b.The molecular
structure
98
MW, the type & the
number of subunits,
The physical-chemical
properties
99
100
estimate the pathologic
changes of the tissue
• apart from the active site
the other structure
concern the catalytic
activity
101
allosteric enzyme
a.concept
b.allosteric activator
allosteric inhibitor
102
c. the characteristics of the
molecular structure
a) oligomers
b) binding site,catalytic site
103
c) the position of the
metabolic pathway
104
substrate intermediate product
Allosteric enzyme
feedback
105
d) the curve of
substrate concentration
106
107
e) Biologic significant
Modification enzyme
a) concept
b) instance
108
glucogen glucogen + glucose
(Gn) (Gn-1)
phosphorylase
Active(+P)
Inactive(-p)
109
6. Enzyme classification &
nomenclature
7.the mechanism of enzyme
action
110
• Formation of an enzyme-
substrate complex is the
first step in enzymatic
catalysis
111
transition state
Binding energy
112
113
E+S ES ES’ E+P unstable
• decrease the activation
energies of reaction
114
active molecule
active energies
115
116
117
• the theory of the the
transition state
• Special catalytic group
contribute to catalysis
118
119
9.the kinetics of enzyme-
catalyzed reaction
120
• The facts affected the rate of
enzyme-catalyzed reaction
substrate
enzyme
temperature
pH
activator
inhibitor
121
• how to mensurate the
rate of the enzyme-
catalyzed reaction
122
• the precondition of the
research the enzyme-
catalyzed reaction
The initial velocity of
the reaction
123
1) Enzyme concentration
affects the rate of enzyme-
catalyzed reaction
v=k[E]
[E]
v
124
2) substrate concentration
affects the rate of enzyme-
catalyzed reaction
125
• 1913 Michaelis Menten
126
[S ]lower [S] vo vo=-d[S]/dt =K[S]
First order
[S] higher [S] v o vo=-d[S]/dt =K[S]
First order
[S] saturated
[S] vo
unchanged
vo=-d[S]/dt =K Zero
order
• rectangular hyperbolic plot
127
128
• Michaelis-Menten
equation:
E + S <---> ES EP E + P
K1
K2
K3
• Michaelis-Menten constant (Km)
129
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
130
• the definition of Km
• the significance of Km
131
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] )
132
K1 K3
E + S ES E + P
K2
( K2 + K3 ) / K1 = Km
Km = K2 / K1 = [ E ] [ S ] / [ ES]
If K2 >>K3
here , Km defined as dissociation constant
133
• the mensuration of the
Km & Vmax
double reciprocal plot
(lineweaver-burk plot)
134
V0= Vmax [S] / ( Km + [S] )
1/v0 =Km
Vmax +
1
Vmax[S]
1
135
136
3) enzyme activity is affected
by temperature
• optimum temperature
137
v
T
optimum temperature
138
139
4) enzyme activity is affected
by pH
• optimum pH
140
141
5) enzyme activity is
affected by activators
• essential activators
• nonessential activators
142
6) enzyme activity is
affected by inhibitors
• irreversible inhibition
specific
143
144
acetylcholine estarase
Acetylcholine acetic acid + choline
145
146
nonspecific
147
Leurisle gas & anti
leurisle gas
148
SH Cl
E + AS-CH=CHCl
SH Cl
S
E AS-CH=CHCl + 2HCl
S
+ SH
OH-CH2-CH-SH E + OH-CH2-CH-S
CH2-SH SH AS-CH=CHCL
CH2-S
149
• reversible inhibition
Competitive inhibition
150
151
characteristics
instance
K1 K3 E+S ES E+P
+ K2
I
EI
Vo Km Vm unchanged
152
153
double reciprocal plot
154
Clinical application
155
angiotensin convert enzyme (ACE)
angiotension angiotensinⅠ Ⅱ
156
noncompetitive inhibition
characteristics
157
158
K1 K3 E+S ES E+P + K2 + I I
EI+S ESI
V , Km unchanged , Vm
159
160
uncompetitive inhibition
characteristics
161
162
K1 K3
E+S ES E+P K2 + I
ESI
V , Km , Vm
163
164
Inhibitor
TypeBinding Site on Enzyme Kinetic effect
Competitive
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.
Noncompetitive
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.
Uncompetitive
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.
165
characteristics
166
10 Enzyme activity detecting
& enzymatic unit
In disease diagnosis
In disease treatments
167
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) various liver disease
LDH ,CK myocardial infarction
lipase Acute pancreatitis
Phosphatase ,acid Metastatic carcinoma of the prostate