Enzyme and Catalysis

Xin Chen ( 陳新 )
xchen@nhri.org.tw

國家衛生研究院 ( 國衛院 )
生物技術與藥物研究組 ( 生藥組 )
   
 Enzyme’s Three Faces:

Catalytic face

Regulatory face

Social face

Arthur Kornberg
Nature Structural and Molecular Biology 11:493

   
Classes of Proteases:

Serine Protease (Chymotrypsin, Trypsin, Subtilisin, DPP-IV etc)

Cysteine Protease (Papain, Cathepsins, Caspases,

viral proteases)

Aspartyl Protease (HIV protease)

Metalloprotease (MMP)

   
 Enzyme and Catalysis

• Catalytic Mechanism;
• Protease Activity Profiling
• Identification of in vivo substrate;
• Substrate Profiling;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzymes as target.

   
Catalytic Mechanism

Proteases:

Hydrolysis of a peptide bond by addition of a

water molecule;
Facilitating a difficult reaction

   
 Essentials:

1. Catalytic Triad (Dyad);

2. Tetrahedral Oxyanion Hole
3. Substrate Specificity

   
Examples:

Chymotrypsin

Prolyl Dipeptidase IV
(DPP-IV)
   
 Serine Protease

Serine residue: Extraordinarily reactive Ser residue

   
 Catalytic Triad
Ser-His-Asp

   
Site-Directed Mutagenesis at Triad and Oxyanion Hole

   
 Tetrahedral Intermediate and Oxyanion Hole

   
DPP-IV (Dipeptidyl Peptidase IV, CD26)(EC 3.4.14.5)

Serine protease: Ser 630

Substrate Specificity:
Prolyl dipeptidase

X-Pro--X-X-X-
Rasmussen et al (2003) X-Ala--X-X-X-

   
 Active Site of DPP-IV

oxyanion Triad

Dipeptidase

Catalytic triad: Ser630-Asp708-His740

   
 Y547 is Essential for Catalysis

Km kcat /Km
kcat
( (ms -1
(s )
-1
mM) mM -1)

rDPP4 279 1.43 195

Y547F 5.56 44 0.128

Bjelke et al. (2004) JBC

   
Substrate Specificity:

Cleave selectively on
the peptide bonds
after Trp, Tyr, Phe
and Met

   
 Enzyme + Substrate --> Reaction

Substrate Specificity
• Increase Catalytic Efficiency
• Stabilize the transition state
• Induced fit

   
Identify Residues Important for Catalysis:

Site-Directed Mutagenesis

Co-Crystal Structure with Inhibitor Mimicking Tetrahedral State

Kinetic Constant Determination

Triad and Oxyanion Hole

   
 Enzyme and Catalysis

• Catalytic Mechanism;
• Protease Activity Profiling
• Substrate Profiling;
• Identification of in vivo substrate;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzyme as target.

   
 Profiling Protease Activity

Tools

Question Answered

   
 Small Molecule Substrate Reporters

   
    Han et al. (2005) Biochemistry
 Inhibitor Screening Platform

Abz-FRLKGGAPIKGV-EDDNP
(FRET: Fluorescence Resonance Energy Transfer)

Fluorescence Quenches

Ex/Em = 320/420 nm +PLP2

Abz-FRLKGG
APIKGV-EDDNP
  Fluorescence Intensity
  Increases!
 Protein-Based Reporter--FRET in vivo

caspase
BFP-DEVD-GFP BFP + GFP
   
 In Vivo Imaging

    Rehm et al. (2002) JBC

 Bioluminescent reporters-In vivo imaging

ER=Estrogen receptor
    Laxman et al. (2002) PNAS
 Noninvasive real time imaging of apoptosis

B: Glioma treated by TRAIL.

    Laxman et al. (2002) PNAS
 Activity-Based Probe (ABP)

Peptides
Alkyl
cross linker

Epoxide
Vinyl sulfates I
125

AOMK Fluorophores
FPs (DFP) Biotin
Hydroxamates HA
   
 Visualization of Protease Activity

   
   
 Summary: Profiling Protease Activity

3. Small molecule substrate reporter

4. Protein-based substrate reporter
5. Bioluminescent substrate reporter
6. Activity-based probe

   
 What else to do with all these tools?

   
 Cancer Cell (2004) 5:443-453.
   
 Microarray Study-mRNA Level (Affymetrix)

N: normal
H: hyperplastic
A: angiogenic
T: tumor

Joyce et al. (2004) Cancer Cell

   
 ABP targeting to cysteine protease

Biotin-tag

fluorophore-tag

   
 ABP Labeled->IP by Biotin ->Run gel (MASS)

Array Data Activity Profiling

   
 Imaging of Cathepsins’ Activity in vivo

Normal

Angiogenic Islets

Tumors

   
In vivo imaging of HPV-Induced Cervical Carcinogenesis

Tumor
Normal +PBS

   
 Chemical Proteomics

   
 Determine/Identify Protease Expression/Activity
in a Cellular Context

   
Probe/Identify Protease Activity in a Cellular Context

   
 ABP Profiling of the Serine Proteases

   
Jessani et al. (2004) PNAS 101:13756
 Activity Correlated with Invasiveness:
Biomarker Discovery

    Jessani et al. (2002) PNAS 99:10335

 Application in Drug Discovery

Inhibitors:
Selectivity
Potency

   
 Biomarker Discovery

   
 Enzyme and Catalysis

• Catalytic Mechanism;
• Protease Activity Profiling
• Identification of in vivo substrate;
• Substrate Profiling;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzyme as target.

   
 Substrate-Protease Interaction

Identify Substrate

Substrate Specificity Profiling

   
Nomenclature for Protease-Substrate Interactions

Substrates: P1, P2, P3..

Enzyme: S1, S2, S3…
Berger and Schechter (1970)
   
 Methods to Determine the Substrate Specificity

3. Molecular Biology

5. Phage display screening

7. Polypeptide coupled with HPLC and MASS

9. Proteomics and MASS

11.Chemical-Based Substrate Profiling

13.Computer-assisted Intelligent guess/

 
Bioinformatics  
  SARS-CoV PLP2  Cleavage Site Prediction
 Cleavage inside the cells

Hartcourt et al (2004) JV
   
 Assays to Determine Substrate Cleavage

+PLP2
FRLKGGAPIKGV FRLKGG + APIKGV

MW MW
=67 584
=58
7 4

677

RP-HPLC Assay
   
MASS Spectrometry
Han et al (2005) Biochemistry
 P1 and P4 Sites are Most Critical

   
Han et al. Biochemistry 2005
 LXGG Motif

   
 PNAS (2006) 103:5717.

   
 Inhibitor Fingerprinting

   Greenbaum et al. (2002) Biol. Chem.

 Analysis of Sub-site Specificity

    Greenbaum et al. (2002) Biol. Chem.

 722 member Ac-Ala-X-X-Arg/Lys-coumarin library

   
Molecular and Cellular Proteomics (2005)
 Substrate Profiling:

High Throughput
High Content
High Technology

   
 PNAS (2004) 101: 6917-6922
   
   
   
    et al PNAS (2004) 101: 6917-6922
Tam
 Discovery of Natural Substrates (II)

   
 PNAS (2004) 101: 11785

   
 Lysate (Freeze and thaw)

Genzyme treated

Looking for Genzyme’s

Substrates Labeled Cy3 or Cy5

2D Differential GE (DIGE)

   
MASS
   
 Green Dots: Potential Substrates
 
Red Dots: Potential  Cleavage Products
 Enzyme and Catalysis

• Catalytic Mechanism;
• Protease Activity Profiling
• Identification of in vivo substrate;
• Substrate Profiling;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzyme as target.

   
 Two Dimerization Interfaces of DPP-IV

C-Terminal Loop

Propeller Loop

From Rasmussen et al.

Nature Structure Biology 2003
   
 DPP-IV is Dimeric in Solution

Recombinant DPP-IV
Human Semen DPP-IV (from baculovirus-infected
Insect cells)

Analytical Ultracentrifugation (AUC)

   
Enzymatic Activity Correlated with Quaternary Structure

DPP-IV

Dimer ==> Monomer

(Low Activity: kcat effect)

Chien et al. JBC 2004;

Chien et al. Biochemistry 2006;
Chen et al., in preparation
   
 B

Site-Directed Mutagenesis of
the Conserved Residues
at Dimer Interface

   
 C-terminus is Highly Conserved among DPPs

*
* Catalytic Triad: Ser630-Asp708-His740

   
Single Site Mutation Disrupts Dimer Formation of DPP-IV

Analytical Ultracentrifugation (AUC)

WT F730A

F713A W734A

V724A Y735A

   
Enzymatic Activity Correlates with Quaternary Structure

Quaternary kcat (s–1) Km (uM) kcat /Km

Structure (s1uM–1)
rDPP-IV Dimer 87 ± 1 90 ± 2 967
F713A Monomer 1.8 ± 1.2 50 ± 2 36
Dimer 48 ± 9 88 ± 1 545
V724A
Monomer 0.5 ± 0.03 76 ± 2 7
Dimer 28 ± 2 56 ± 2 500
F730A
Monomer 0.2 ± 0.01 54 ± 5 4
W734A Monomer 0.3 ± 0.2 90 ± 0.4 3
Y735A Monomer 0.05 ± 0.02 89 ± 1 0.6
Dimer 73±6 79±5 924
H750A
Monomer 1.4±0.3 64±2 22
   
C-terminal Loop, the S1 Site and Triad of DPP-IV

  *  
* Catalytic Triad: Ser630-Asp708-His740
Summary:

1. Dimerization is essential for optimal activity

of DPP-IV;
2. Enzymatic activity correlates with quaternary
structure

Dimer ==> Monomer

(Low Activity)

Chien et al. JBC 2004;

Chien et al. Biochemistry 2006;
  Chen
  et al., in preparation
 Different Interaction Mode at Dimer Interface

DPP8

Dimer ==> Dimer

(Low Activity: kcat and Km effect)

Chen et al. (2004) Prot. Exp. Purif.

Lee et al., (2006) JBC
   
Enzymatic Activities of Full Length DPP-IVs
120

100
100 65
Enzymatic%Activity
80

60
45
40

20 7
0.98
0

Vector WT F713A W734A Y735A

Ectodomain Enzyme Activity 100 3.7 0.3 0.1

    Ongoing study
Transmembrane Domain is a Dimerization Motif?

   
 TM is predicted to be alpha helix
with heptad repeat
Reside 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2
7 8 9
# 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8

W=28 V L L G L L G A A A L V T I I T V P V V L L

COILS f e f g a b c d e f g a b c d e f g a b c d

Paircoil
d e f g a b c d e f g a b c d e f g a b c d
2

   
 Helical Wheel Modeling of TM
   
 Bioluminescence Resonance Energy Transfer
(BRET) Assay

Fluorescence
DeepBlueC Light at 395 nm at 510 nm

RLuc GFP

   
 BRET Assay

160
123
140

120 100

100 90
BRET Ratio (mBU)

80

60
31 33
40
-27
20

-20 WT G13I G14I G14P

-40

TM-Rluc + - + + + +
TM-GFP2 - + + + + +
Rluc - + - - - -
GFP2 + - -  - - -
 
 Three Dimerization Interfaces of DPP-IV

Dimerization is a folding event

  Like a  zipper!
   
 Enzyme and Catalysis

• Catalytic Mechanism;
• Protease Activity Profiling
• Identification of in vivo substrate;
• Substrate Profiling;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzyme as target.

   
 DPP-IV (Dipeptidyl Peptidase IV, CD26)(EC 3.4.14.5)

• Prolyl dipeptidase (Serine protease: Ser 630);

X-Pro--X-X-X-
X-Ala--X-X-X-

7. Exists extra-cellularly;

3. In vivo substrates:

Hormones (GLP-1, GIP, etc);

Chemokines (RANTES, MRC, etc);
Neuropeptides (NPY)

   
DPP-IV, Glucose tolerance and Type II Diabetes

Glucose Homeostasis

Metabolites

DPP-IV Inhibit

GLP-1 Glucose-Dependent Glucose

GIP Insulin Secretion Homeostasis
t1/2=1-2min

   
DPP-IV, Glucose Tolerance and Type II Diabetes

Glucose Homeostasis

Metabolites
Inhibit
DPP4 + Inhibitors

GLP-1 Glucose-Dependent Glucose

GIP Insulin Secretion Homeostasis
t1/2=1-2min

   
DPP-IV, Glucose Tolerance and Type II Diabetes

Glucose Homeostasis

Metabolites
Inhibit
DPP4 + Inhibitors

GLP-1 Glucose-Dependent Glucose

GIP Insulin Secretion Homeostasis
t1/2=1-2min

GLP-1/GIP
Insulin secretion
   
Glucose tolerance
 DPP-IV -- a validated and effective drug target
for type II diabetes

1. Healthy phenotypes with improved glucose tolerance

and insulin sensitivity on DPP-IV knockout mice and rabbits;

• Insulin-sensing hormones GLP-1 and GIP are the in vivo

substrates of DPP-IV;

10. DPP-IV inhibitors are effective to treat Type II diabetes

in animal models and human clinical trials.

   
 Drug Discovery with DPP-IV as the Target

DPP-IV
Rational Design/HTS/VS

Drug compounds

Optimization
Potency
Selectivity FAP, DPP8, DPP9, DPP2 etc

ADEM, Pharmacokinetic study (PK)

Safety and efficacy on animals
  Toxicity, Preclinical
  trial…..
 Human Prolyl Dipeptidases X1-Pro2-XX

From Rosenblum and Kozarich, 2003

   
 Functions of Other DPPs Unknown

FAP

Exoprotease; Biomarker for cancers

Specifically expressed in malignant epithelial
type of cancers and during wound healing

DPP8 and DPP9

Cytosolic enzyme (Abbott et al. 2003).

Upregulation in T-cell activation (Abbott et al. 2003);
Administration of DPP8/9 Inhibitors to
animals results in severe toxicity
(Lankas et al., 2005)
   
 Structural Similarity

DPP-IV

FAP DPPX

   
 Counter-Screening with DPP Family of
Proteases is Essential

   
 Screening Platforms

• Purified Enzymes
Endogenous source (human semen) or recombinant
forms from baculovirus-infected insect cells

• Substrates

For DPP-IV:
Ala-Pro-pNA, Ala-Pro-AMC, Gly-Pro-Luciferin
(Promega)

   
 HTS Platform

Blank Blank

Substrate only Sub.+enzyme only

BPR1G001 1 uM

Blank Blank

Gly-Pro-pNA as substrates

   
Substrate Profiling to Find Optimal Substrate
DPP8

Ala-X-pNA

X-Pro-pNA

    Lee et al. (2006) JBC

 Potency and Selectivity
DPP-IV DPP8 DPP-II FAP
IC50 (nM) IC50 (nM) IC50 (nM) IC50 (nM)
OH

N
N
H O CN
51 14219 >100,000 >100,000
NVP LAF237

NH2 O

F N N
N 37 > 20,000 >20,000 >20,000
F N
CF3
MK-0431

1G-338 54 > 20,000 >20,000 >20,000

1G-328 1.7 2727 >20,000 175

   
 Plasma DPP-IV Inhibition and OGTT
18 mg/kg to mice 10 mg/kg to rats (3 g/kg OGTT PO)

14 Control
LAF-237 180 Control
1G-226 LAF-237
DPP4 Activity of Plasma (mU/ml)

12 1G-250 1G-226
160
10

Blood Glucose (mg/dl)

140
8
120
6

4 100

2 80

0 60
0 1 2 3 4 -40 -20 0 20 40 60 80 100 120
Time (Hr) Time (min)

U.S. Serial No. Filing Date Status

N N 60/551,419 March 9, 2004 Pending
O O 60/617,684 October 12, 2004 Pending
N N
N N
H H O CN
O CN
  1G-226 1G-250   J. Med. Chem. 2006, 49, 373.
Directions of Future Studies on DPP Enzymes

• Catalytic Mechanism;
• Protease Activity Profiling
• Identification of in vivo substrate;
• Substrate Profiling;
• Enzymatic Regulation (Quaternary Structure)
• Drug discovery with Enzyme as target.