Enzyme Behavior

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Lecture 7 Enzyme Behavior Chapter 7 Also Chapter
4 pp. 97-99 104-99
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• Topics to be covered
• Enzyme mechanisms
• Enzyme regulation
• A. Allosteric enzymes
• Hemoglobin
• B. Zymogen activation
• C. Enzyme phosphorylation

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1. Mechanisms of enzyme action.
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• Questions
• Which amino acids are located at the active site
  on an enzyme?
• How do they act to catalyze the reaction?

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Digestive enzymes cleave proteins.
Peptide
A-B-C-D-E-F-G
Cleaves After D.
Trypsin D positively-charged lysine arg
inine
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1. Substrate binds.
Chymotrypsin Dhydrophobic Tryptophan
Phenylalanine
Binding pocket with greasy sides.
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Trypsin D positively-charged lysine
arginine
-
Negative charge at the bottom of the pocket.
Example of a family of enzymes.
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Enzyme families
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• The mechanism Strategy
• Need a reaction that is easy to
• measure.

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Chymotrypsin

• Reaction with a model substrate

Ester
Colored
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• Identify the amino acids at the
• active site that are involved in the
• reaction.
• Use molecules (inhibitors) that
• covalently bind to specific
• amino acids at the active site
• and inhibit the reaction.

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Chymotrypsin

• DIPF inactivates chymotrypsin by reacting with
  serine-195, which must be at the active site

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Chymotrypsin

• TPCK labels histidine-57

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Chymotrypsinbecause Ser-195 and His-57 are
required for activity, they must be close to each
other in the active site
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• in addition to His-57 and Ser-195, Asp-102 is
  also involved in catalysis at the active site

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results of x-ray crystallography show the
definite arrangement of amino acids at the active
site
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(No Transcript)
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Mechanism of hydrolysis by chymotrypsin Hydrolys
is add water take the molecule apart
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His 57
Ser 195
OH
N
N
O
C-N-R2
R1
H
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His 57
Ser 195
OH
N
N
O
C-N-R2
R1
H
Nucleophilic attack
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His 57
Ser 195
OH
N
N
O
C-N-R2
R1
H
Serine proteases
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His 57
Ser 195

O H
N
N
-
O
C-N-R2
R1
H
Tetrahedral intermediate
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His 57
Ser 195

O H
N
N
-
O
C-N-R2
R1
H
Tetrahedral intermediate
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His 57
Ser 195

O
N
N
O
C
R1
NH2 R
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His 57
Ser 195

O
N
N
H O H
O
C
R1
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His 57
Ser 195


O
N
N
H O H
-O
C
R1
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His 57
Ser 195
O-H
N
N

O
C
OH
R1
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2. Covalent modification of enzymes. Phosphorylat
ion of OH groups on serine, threonine tyrosine
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Phosphorylase breaks down glycogen muscle
liver
Inactive
Active
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Zymogen an inactive precursor of an enzyme
cleavage of one or more covalent bonds transforms
it into the active enzyme
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Chymotrypsin secreted by thepancreas as the
inactive zymogenchymotrypsinogen.
Protease action converts it to the active form
chymotrypsin.
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Chymotrypsinogen (inactive)
p-chymotrypsin (actuve)
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The cleavage changes the secondary and tertiary
structure which changes the structure of the
active site.