Proteases

Dr. Jessica Bell
Davies Laboratory
NIDDK/NIH 
For the 
  University of Richmond
 
What do proteases do?
H
H O H +
3 HN C COO­
+
HN
3 C C N C COO­ + H2O R1 + H
R1 H R2 +
3 HN C COO­
R2

∆Go for the rxn is ­2kcal/mol
But…
Uncatalyzed rxn at neutral pH,  Catalyzed rxn (chymotrypsin) at 
37°C: 1 X 10­10 /sec neutral pH, 37°C: 100/sec

Conditions for chemically catalyzed 
reaction:
   
Koshland, D. (1996) J. Cell. 
24hrs. @ 6M HCl, 110°C Comp. Phys. Suppl. 1 43:217.
 Two types of cleavages
endopeptidase

exopeptidase

Same rxn, Four mechanisms
Named for residue/group in active site of enzyme essential for 
most effective catalysis
Serine ­OH
Cysteine/Thiol ­SH
Acid/Aspartic ­COO­
   
Metallo Zn2+
Mechanistic Sets of Proteases

set feature inhibitor examples function

Serine protease active site serine fluorophosphates trypsin digestion

H57, D102, S195 thrombin blood coagulation
plasmin lysis of blood clots
coccoonase mechanical
subtilisin digestion
acrosin sperm penetration

Cysteine protease active site cysteine iodoacetate papain digestion

C25, H159, N175 strept. proteinase digestion
cathepsin B intracell. digestion 

Acid protease acidic pH optimum diazoketones pepsin digestion

D32, D215 chymosin milk coagulation

Metalloproteases Zn2+, E270 o­phenanthroline carboxypeptidase digestion

Zn2+, Ca2+ o­phenanthroline thermolysin  digestion
E143, H231
   
 Adhesion
Secretion P. gingivalis protease
signal peptidases
Immune 
Response
Development T­cell protease
snake
Blood pressure 
6 Broad Categories
Digestion regulation
renin Function           Protease
trypsin
Coagulation
thrombin Nutrition   trypsin, subtilisin, α­lytic 
Complement    protease
Fixation Cell fusion
CI protease hemaglutinase Invasion   matrix metallo proteases

Tumor  Evasion   IgA protease

Reproduction 
and  Invasion Adhesion   P. gingivalis protease
collagenase
Fertilization
acronase
Processing  signal peptidase, viral 
  proteases, proteosome
Signaling   caspases, granzymes
Fibrinolysis Pain Sensing
tissue  kallikrein
plasminogen 
actvator

Animal Virus 
Hormone  Replication
HIV protease
Processing
Kex 2
   
Serine Protease Mechanism – The players

    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
Serine Protease Mechanism – Oxyanion Hole

    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
    Adapted from Voet and Voet 
(1995) Biochemistry, 2nd ed. John 
Wiley and Sons, Inc. New York.
Serine inhibitors

CH3
O O
S NH CH C CH2 Cl
O CH2
Peptide bond mimic

Chloro­methyl ketone [CMK]
TPCK
(L­1­Chloro­3­[4­tosylamido]­4­phenyl­2­butanone)
   
Serine inhibitors

CH3 F CH3
CH O P O CH

CH3 O CH3

DFP
Diisopropyl fluorophosphate

   
Divergent vs. Convergent Evolution

Catalytic Triad Conserved

Trypsin Elastase Subtilisin

   
Same Fold
 Serpins
Serine protease 
inhibitors
Irreversible
Disruption of 3º 
structure

   
 Ecotin
Serine Protease Inhibitor
Unknown function
Dimeric
1° and 2° binding sites
Cleaved

   
Cysteine protease mechanism
Michaelis Complex Tetrahedral intermediate I
159 159
25
25
S
+
N N: H N N H S
H H O­
O HN
HN

P1
P1

159 159

25 25
N + N H S N N: S
H O­ H O
NH2
O
    NH2
H
Tetrahedral intermediate II P1 H2O Acyl Intermediate P1
Cysteine protease mechanism
Michaelis Complex Tetrahedral intermediate I
159 159
25
25
S
+
N N: H N N H S
H H O­
O HN
HN

P1
P1
Covalent 
Intermediate
No Asp102 
159
equivalent 159

25 25
N + N H S N N: S
H O­ H O
NH2
O
    NH2
H
Tetrahedral intermediate II P1 H2O Acyl Intermediate P1
Cysteine protease inhibitors

159
25

S
N N
:
H
H

I
O
CH2 C OH

Iodoacetic acid

E­64
(2S,3S)­3­(N­(1S)­1­[N­
(4guanidinobutyl)carbamoyl]3methylbutyl)carbamoyl) 
oxirane­2­carboxylic acid

   
 Cystatin Superfamily
Cysteine protease inhibitors
Non­canonical binding

   
Acid protease mechanism
Michaelis complex
H P1 P1’ N H P1 N P1’
O O
H H
O O
O H O
H
O H H
O
­O O O O
­ ­

Asp25 Asp25’ Asp25 Asp25’

P1 H P1’ P1 P1’
H O
N
O
N
H
O­
H H
O
O O
H
O O
O O­ O O
­H ­
H
   
Asp25 Asp25’ Asp25 Tetrahedral  Asp25’
intermediate
Acid protease mechanism
Michaelis complex
H P1 P1’ N H P1 N P1’
O O
H H
O O
O H O
H
O H H
O
­O O O O
­ ­

Asp25 Asp25’ Asp25 Asp25’

No covalent 
intermediate
Activated water
P1 H P1’ P1 P1’
H O
N
O
N
H H H
O O­
O O
H
O O
O O­ O O
­H ­
H
   
Asp25 Asp25’ Asp25 Tetrahedral  Asp25’
intermediate
Acid protease inhibitors

   
Indinavir, Roche
 CH3 O
H

RHN
N
N
NHR’ HIV Protease Substrate
O O

O
H

   
Reiling, K. K. et al. Biochemistry (2002) 41:4582­94.
Movie of Multi­drug resistant HIV Models:

www.ucsf.edu

Click on A­Z listings
Under C find Craik, Charles
Within the Craik website there is section 
entitled movies
Enjoy!

   
 Pepsin

HIV 
Protease

   
Metallo protease mechanism
Glu
H
­
His Zn2+ O
His O
Glu His Glu
H His H O
His Zn2+ O
O
Zn2+

H H
O
His N
P1 P1’

His
His Glu His
­ His Glu
­O
Zn2+
Zn2+
O
­
His O
O
His Glu H
O O O
P1
N H
P1 O­ Zn2+
­
­O P1’
O O
O

P1
H
   
H
N
P1’
Metallo protease mechanism
Glu
H
­
His Zn2+ O
His O
Glu His Glu
H His H O
His Zn2+ O
O
Zn2+

H H
O
His
No covalent 
N
P1 P1’
intermediate

His
His
Glu
Activated water His
­ His Glu
­O
Zn2+
Zn2+
O
­
His O
O
His Glu H
O O O
P1
N H
P1 O­ Zn2+
­
­O P1’
O O
O

P1
H
   
H
N
P1’
 H2N­Asp­Arg­Val­Tyr­Ile­Pro­Phe­His­Leu­Co2H
Proangiotensin

H2N­Asp­Arg­Val­Tyr­Ile­Pro­Phe­Co2H
Angiotensin

Zn2+ A
H

H O H O H O H2N
NH C C N C C N C C ­ + C NH Arg
R2 H R1 H R1 O H2N

carboxy­di­peptidase active site

Zn2+ A
H

S H O H O H2N
CH2 C C N C C ­ + C NH Arg
CH3 O H2N
   
Captopril
 Thermolysin

Carboxypeptidase A

   
Synopsis of Protease Mechanisms
Serine
Ser­His Asp Catalytic Triad
covalent intermediate
Cysteine
Cys­His
covalent intermediate
Acid 
Asp­Asp
Activated water
no covalent intermediate
Metallo
Zn2+ or equivalent­Glu

 
Activated Water
 

no covalent intermediate
How Proteases Order Off the Menu
P2 P1 P1’ P2’
OH
H O CH3 H O
N N
Peptide
N N N
H O H O H

Scissile
Bond

NH3+ OH
Subsite of 
Protease

   
S2 S1 S1’ S2’
Substrate Selection within One Tertiary Fold

   
Methods to Determine Specificity

1> Synthesis of short peptides [15 to 20a.a.], 
check for cleavage with PAGE

2> Phage display of short peptides

3> Positional scanning synthetic 
combinatorial libraries [PS­SCL]
   
 X O X O H
H H
N
N N

O
N N
H O X H O X

7­amino­4­methyl 
coumarin

A R N D E Q G H I L
Ac­XXXO­AMC
K F P S T W Y V m
A R N D E Q G H I L
Ac­XXOX­AMC
K F P S T W Y V m
A R N D E Q G H I L
Ac­XOXX­AMC
K F P S T W Y V m
A R N D E Q G H I L
Ac­OXXX­AMC
K F P S T W Y V m
    Harris J. L. et al. Rapid and general profiling of 
protease specificity by using combinatorial fluorogenic 
substrate libraries. PNAS (2000) 97:7754­9.
400.0 0.06

0.058
300.0 0.056

0.054
200.0 0.052

0.05
100.0 0.048

0.046
0.0 0.044
A R N D Q E G H I L K F P S T WY V mM A R N D Q E G H I L K M m F P S T W Y V

P4 H O P2 H O
N N

N N
H O P3 H O P1

500.0 200.0

400.0
150.0

300.0

100.0
200.0

50.0
100.0

0.0
   
0.0
A R N D Q E G H I L K F P S T W Y V mM A R N D Q E G H I L K F P S T W Y V mM
Regulation of Proteases – A Few Examples
Zymogens
Pro­peptide that must be cleaved before protease becomes fully active
Trypsinogen
1 16
Enteropeptidase

1 15
Trypsin
16

Zymogen form has distorted oxyanion hole and substrate binding pocket

Compartmentalization
Macromolecular Inhibitors
Host and non­host

   
 Cytotoxic Lymphocytes

   
Molecular Biology of the Cell, Garland
Cytotoxic T Lymphocyte Apoptotic Pathway

Cytotoxic T 
lymphocyte

Granzymes

Perforin Ca2+
Ca 2+
Ca2+

3 Fas
Ca2+
GrnB GrnA
MPR?
DD
FADD cleave  serpins Nuclease
DED pro­caspases ?
Mito.

apoptosis Bcl­2 Single stranded 

aggregrates pro­caspase  breaks in DNA
8, intermolecular cleavage 
to caspase 8, activation of 
effector caspases [3, 6, 7],  nucleus
  apoptosis  
Granzymes: Lymphocyte Serine Proteases
Name Activity Predicted P1  MW
cleavage site                     
A Trypsin­like                 R/K 60 (Dimer)
B Asp­ase                  D/E 35
C Unknown         N/S 27
D Unknown         F/L 35­50
E Unknown         F/L 35­45
F Unknown         F/L 35­40
G Unknown         F/L
H Chymase                  F
I Unknown
J Unknown

  K Trypsin­like   30
M Met­ase   M/L/nor­L 30
 Granzyme Structure

   
Waugh et al. (2000) Nat. Struct. Biol. 7:762­765
Granzyme A, Proposed Dimeric Structure

   
 Granzyme A: Substrate Specificity and 
Macromolecule Substrates
Substrate     Sequence
P4 P3 P2 P1
FLUOROGENIC LIBRARIES V/I G/A/S N R
PIL-1β D A P V R S L N C T
THROMBIN RECEPTOR T L D P R S F L L R
HISTONE H1 K L G L K S L V S K
HISTONE H2b A P A P K K G S K K
SET Q T Q N K A S R K R
LAMIN B V T V S R A S S S R

   
 Chasing the Crystals

   
 Macromolecular Inhibition of 
Granzyme A
1.2

1
Control

0.8 mM84R Eco
mOD/min @ 405nm

dM84R Eco
0.6

Tryp. Inh.
0.4

0.2

0
  0 0.05   5 50
[Inhibitor], µM
 Potential Effects of Oligomer on 
Macromolecular Inhibitors

  grnA  
 Potential Effects of Oligomer on 
Macromolecular Inhibitors

grnB:dEcotin

   
 Potential Effects of Oligomer on 
Macromolecular Inhibitors

mEcotin

   
Small Molecule Inhibitor of Granzyme A
0.8

0.7
O O

0.6 N C C N C C N C C CH2Cl
O
mOD/minute @405nm

0.5

0.4

0.3

0.2

0.1

0
  0 50   100 150 200
[Inhibitor], nM
Crystallization
Previous conditions:
0.1M Citrate, pH 5.6, 20% peg 4K, 20% Isopropanol
New Conditions:
4M NaFormate
0.1M Citrate, pH 5.6, 20­30% peg4K, 0.2M AmAcetate
0.1M Cacodylate, pH 6.5, 15­20% peg4K, 0.2M AmSO4
0.1M Tris, pH8.5, 13­18% peg4K, 0.2M LiSO4

   
Diffraction!!!

   
 Substrate Selectivity

   
 Granzyme A: Human and Mouse
Human MRNSYRFLAS SLSVVVSLLL IPEDVCEKII GGNEVTPHSR PYMVLLSLDR
Mouse MRNASGPRGP SLATLLFLLL IPEGGCERII GGDTVVPHSR PYMALLKLSS

Human KTICAGALIA KDWVLTAAHC NLNKRSQVIL GAHSITREEP TKQIMLVKKE

Mouse NTICAGALIE KNWVLTAAHC NVGKRSKFIL GAHSINK-EP EQQILTVKKA
#
Human FPYPCYDPAT REGDLKLLQL TEKAKINKYV TILHLPKKGD DVKPGTMCQV
Mouse FPYPCYDETT REGDLQLVRL KKKATVNRNV AILHLPKKGD DVKPGTRCRV
#
Human AGWGRTHNSA SWSDTLREVN ITIIDRKVCN DRNHYNFNPV IGMNMVCAGS
Mouse AGWGRFGNKS APSETLREVN ITVIDRKICN DEKHYNFHPV IGLNMICAGD

Human LRGGRDSCNG DSGSPLLCEG VFRGVTSFGL ENKCGDPRGP GVYILLSKKH

Mouse LRGGKDSCNG DSGSPLLCDG ILRGITSFG- GEKCGDRRWP GVYTFLSDKH
# * *

68% Identical!
Human LNWIIMTIKG AV
Mouse LNWIKKIMKG SV

P4 P3 P2 P1
Human           V/I    G/A/S      N  R
  Mouse G        F/Y
   F  R
Substrate Specificity of Granzyme A Species

H57
D102

R99
P2
P1 S195
P4 P3

D189

   
Substrate Specificity of Granzyme A Species

P4

W224

   
Substrate Specificity of Granzyme A Species

P4

W224

   
Substrate Specificity of Granzyme A Species

   
 Native Human GrA
0.06

0.05
Relative Fluorescence Units

Human Mouse
0.04

0.03

0.02
P2      N      F
0.01

0
A R N D Q E G H I L K F P S T W Y V n
P1-Arg PS-SCL of Human GrA - P3
Amino Acid

0.06

0.05
Relative Fluorescence Units

0.04

0.03
P3  G/A/S    F/Y
0.02

0.01

0
A R N D Q E G H I L K F P S T W Y V n
P1-Arg PS-SCL of Native Human GrA - P4
Amino Acid

0.06

0.05
Relative Fluorescence Units

0.04

0.03
P4     V/L     G
0.02

0.01
   
0
A R N D Q E G H I L K F P S T W Y V n
 H ­> M GrA
0.14

0.12

0.1

0.08 Human Mouse

0.06

0.04 P2      N      F

0.02

0
A R N D Q E G H I L K F P S T W Y V n

0.14

0.12

0.1

0.08

0.06
P3  G/A/S    F/Y
0.04

0.02

0
A R N D Q E G H I L K F P S T W Y V n

0.14

0.12

0.1

0.08

0.06
P4     V/L     G
0.04

   
0.02

0
A R N D Q E G H I L K F P S T W Y V n
Conclusions: Mutational Studies

The residues identified from the model of mouse granzyme A [∆
L201, G202, E203, W211] when mutated into the equivalent 
positions of the human homologue:
1> switch the substrate specificity at the P3 position,
2> increase the preference for small residues [A/G] over 
     branched residues [I/V] at the P4 position and
3> broaden residue selection at the P2 position.  

   
 C. S. Craik
Craik Lab Members
Granzyme A R. J. Fletterick
Sandy Waugh  Fletterick Lab Members
Sami Mahrus
Carly Klein
MT­SP1
Jeonghoon Sun
ALS 8.3.1
Ami Bhatt James Holton

The Chemists
Amy Barrios
Alan Marnett
  NIH: The $$$ people