REGULATORY ENZYME

(ENZYMATIC REGULATION)

(WEEK 6)
REGULATORY ENZYMES (Protein-level bio-regulation)

Reversible allosteric regulation

(Modulator: activator, inhibitor)

Reversible covalent modification

Regulated by Other Protein

reversible protein binding

irreversible proteolytic cleavage

 REGULATORY ENZYMES (Protein-level bio-regulation)
Multiple subunit
Active site regulatory site
20.1 Type of Regulations
20.1.1 Reversible allosteric regulation modulator
inhibitor (I) E1 threonine
activator (A) dehydratase
substrat = modulator homothropic
substrat modulator heterothropic E2

E3
feedback
inhibition
E4

E5

20.1.2 Reversible covalent modification

20.1.3 Regulated by Other Protein
reversible protein binding
irreversible proteolytic cleavage
 20.1.2 Reversible Covalent Modications

R groups of specific Ser residues

Phosphorylase b
(inactive)

Phosphorylase Phosphorylase
phosphatase kinase

Phosphorylase a (active)
 Examples of Reversible Covalent Modications
Covalent modification Modified aa
O -

ATP ADP Glycogen phosphorylase, Tyr, Ser,

Phosphorylation: E E P O-
protein kinase, etc. Thr, His
O
NH2

N
O N
ATP PPi
Adenylylation: E E O P O N N
Tyr
O
O- H H
H H
OH H
O

NH
O
UTP PPi Tyr
Uridylylation: E E O P O
O N O
O- H H
H H
OH OH
-Cholera toxin specific G protein massive loss of body fluid death Tyr, Gln,
ADP-Ribosylation: -Diphteria toxin inhibits elongation factor 2
-Dinitrogenase reduktase nitrogen fixation NH2
N
Cys,
E E O O N
diphthamide
NAD nicotinamide O O P O P O O N N
H H O- O- H H
H
H
H
OH
H
OH H
H
modified His

S-adenosyl- S-adenosyl-
methionine homocysteine Methyl-accepting Glu
chemotaxis protein
Methylation: E E-CH3 of bacteria
 20.1.3 Regulation by Other Protein
-reversible protein binding
Epinephrine receptor
Epinephrine Plasma membrane Extracellular space

Activated Cytosol
G protein adenylate cyclase
ATP cAMP (2nd messenger)
Active glycogen synthase
Inactive protein kinase
Active protein kinase Inactive glycogen
(phosphorylated)
Inactive phosphorylase kinase synthase
Active (phosphorylated) phosphorylase kinase

Inactive glycogen phosphorylase

Active (phosphorylated) glycogen phosphorylase

Glycogen
Glucose phosphate Glucose
 20.1.3 Regulation by Other Protein
-irreversible proteolytic cleavage Chymotrypsin activation
Trypsin activation Chymotrypsinogen inactive
1 245
Trypsinogen (inactive)

1 6 7 Trypsin
245
Val-(Asp) 4-Lys-Ile -chymotrypsin active
enteropeptidase 1 15 16
245
Arg Ile
Chymotrypsin
Val-(Asp)4-Lys
Ser14-Arg15
Trypsin (active) Thr147-Asn148
7 245 -chymotrypsin active
Ile
1 13 16 146 149
245
Leu Ile Tyr Ala
A B C
 20.1.3 Regulation by Other Protein
-irreversible proteolytic cleavage Chymotrypsin activation
Trypsin activation Chymotrypsinogen inactive
1 245
Trypsinogen (inactive)

1 6 7 Trypsin
245
Val-(Asp) 4-Lys-Ile -chymotrypsin active
enteropeptidase 1 15 16
245
Arg Ile
Chymotrypsin
Val-(Asp)4-Lys
Ser14-Arg15
Trypsin (active) Thr147-Asn148
7 245 -chymotrypsin active
Ile
1 13 16 146 149
245
Leu Ile Tyr Ala
A B C
 20.1.3 Regulation by Other Protein
-irreversible proteolytic cleavage
Proinsulin
+
H3N -CHAIN C-CHAIN

Signal
peptide -
OOC A-CHAIN
removal

Preproinsulin Proteolytic cleavages and

formation of disulfide bonds

Insulin
+
H3N -CHAIN COO-
=S

+ COO-
Disulfida bridges H3N A-CHAIN
 Regulation of Enzyme Activity
Inhibitor Proteolysis
or proteolysis
o I I x x o
S I I S

inhibitor

Feedback regulation Phosophorylation

o R x x P o
S R S P
(+)
regulator
effector phosphorylation
Signal transduction
A or A
x o
Regulatory
(-) subunit
S
+ cAMP or
calmodulin
cAMP Controls Activity of Protein Kinase A

Regulatory A
A Active kinase
subunits A cAMP
A
A C
R C R
A
A
R C R C
Catalytic A
subunits

Nucleus CREB
Activation C
P
Gene
CREB expression
DNA ON
 Signal Transduction Network (Ras vs. P53)
Signal
Cell function are controlled by protein interactions
Receptor
Cell membrane
Cytosol Ras
Regulator protein
Signal protein Effector
Transcription
enzyme
Ribosome
Inhibitor
Apoptosis
Nucleus

P53 E2F
mRNA Transcription
factor Target gene

Transcription
mRNA Cell division ON
 XII ENZYME ANALYSIS

- Protein analysis: Isolation and Purification

Spectrophotometri
Electrophoresis
Chromatography
Affinity-based Method
X-Ray Crystallography
Sequencing
Synthesis

- Activity analysis

- Inhibition analysis

- Etc.
 XIII ENZYME APPLICATION
Enzyme Source Application
Alcohol dehydrogenase Saccharomyces cereviceae Alcohol testing
Aspergilus oryzae Food and beverage
-Amylase Bacillus substilis industry

Aspergillus niger
Amyloglukosidase Glucose syrupe
Aspergillus oryzae
Cellulase Trichoderma viride Drain cleaner
B. coagulans
Glucose Isomerase Streptomyces, sp Isomerisation of
glucose to fructose
Streptomyces albus
Invertase Saccharomyces cereviceae Invert sugar

Escherichia coli Semisynthetic

Penicilin Acylase penicillin (6-APA) raw
Bacillus substilis material

Dextranase Penicillium funicolosum Polysaccharide

hydrolysis

-Galactosidase (Lactase) Escherichia coli Lactose hydrolysis

 EXAMPLES OF COMMERCIAL ENZYME IN INDUSTRIAL APPLICATION

Enzyme Commercial name Application

Bacterial -Amylase Aquazim Textil, alcohol, sugar, detergent

Amyloglucosidase AMG Alcohol, bread, starch

Cellulase Celluclast Alcohol, juice, wine

Dextranase DN 25L Sugar

Glucose isomerase Sweetgyme Starch

Lactase Lactogyn Milk industry

Pectinase Pactinex netragin Food processing

Bacterial proteinase xxAlealase, Savinase Detergent, leather, food