Download Anne-Marie Lambeir

ITC for carboxypeptidases:
method comparison
Anne-Marie Lambeir
Paris, 26 September 2016
Why use ITC for carboxypeptidase research?
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Why use ITC for carboxypeptidase research?
aminopeptidase
+ H2O
Arg-4-methoxy-2-naphthylamine
3
Arginine +
Why use ITC for carboxypeptidase research?
Proline specific endopeptidase
+ H2O
Z-Gly-Pro-p-nitroanilide
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Z-Gly-Pro +
Why use ITC for carboxypeptidase research?
Basic carboxypeptidase
O
NH
OH
+ H2O
H C
3
NH
+
HN
O
2
NH
OH
NH
O
Benzyl-Ala-Arg
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2
Why use ITC for carboxypeptidase research?
 Carboxypeptidases hydrolyse the peptide bond preceding
the C-terminal amino acid
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Why use ITC for carboxypeptidase research?
 Carboxypeptidases hydrolyse the peptide bond preceding
the C-terminal amino acid
 Their specificity is determined by the P’1 residue
P1
P’1
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Why use ITC for carboxypeptidase research?
 Carboxypeptidases hydrolyse the peptide bond preceding
the C-terminal amino acid
 Their specificity is determined by the P’1 residue
 They recognize the terminal carboxyl group
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Why use ITC for carboxypeptidase research?
 Carboxypeptidases hydrolyse the peptide bond preceding
the C-terminal amino acid
 Their specificity is determined by the P’1 residue
 They recognize the terminal carboxyl group
 Enzymatic assays need to respect this
 No modified amino acids
 No chromogenic or fluorigenic leaving groups
 No C-terminal modifications
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Carboxypeptidase M (CPM) and prolyl
carboxypeptidase (PRCP)
 Carboxypeptidases fine-tune or inactivate peptides
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Carboxypeptidase M (CPM) and prolyl
carboxypeptidase (PRCP)
 Carboxypeptidases fine-tune or inactivate peptides
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
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Carboxypeptidase M (CPM) and prolyl
carboxypeptidase (PRCP)
 Carboxypeptidases fine-tune or inactivate peptides
 CPM modulates the signal of bradykinin and anaphyllatoxins
 Inflammation
 Blood pressure
 Shock
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CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Carboxypeptidase M (CPM) and prolyl
carboxypeptidase (PRCP)
 Carboxypeptidases fine-tune or inactivate peptides
 CPM modulates the signal of bradykinin and anaphyllatoxins
 Inflammation
 Blood pressure
CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
 Shock
 PRCP modulates the signals of anorexigenic peptides,
angiotensins and des-Arg-bradykinin
 Body weight control
 Blood pressure
 Inflammation
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PRCP
Carboxypeptidase M (CPM) and prolyl
carboxypeptidase (PRCP)
 Carboxypeptidases fine-tune or inactivate peptides
 CPM modulates the signal of bradykinin and anaphyllatoxins
 Inflammation
 Blood pressure
CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
 Shock
 PRCP modulates the signals of anorexigenic peptides,
angiotensins and des-Arg-bradykinin
 Body weight control
 Blood pressure
 Inflammation
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PRCP
Methods used for CPM
Substrate
Analyte
Equipment
Issues
Bz-Gly-Arg
Bz-Glycine
HPLC
High Km
Ethylacetate extraction
Internal standard
X-Arg
Arginine
HPLC
o-phtalaldehyde
derivatisation
Dansyl-Ala-Arg*
Dansyl-Ala
fluorimeter
Chloroform extraction
Furfurylacryloyl-Lys Furfurylacryloyl Spectrophotometer Low sensitivity
340 nm
X-Arg
Arginine
Microtiterplate
Reader – 340 nm
Coupled enzymes
Expensive reagents
* Only substrate suitable for cell lysates and other complex biological samples
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Arginine kinase coupled assay
CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
CPM
X-arginine → X + arginine
pyruvate
+
+
ATP
AK
NADH
LDH
PK
ADP + phospho-arginine
PEP
lactate + NAD+
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Arginine kinase coupled assay
CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
17 µM
34 µM
1250 µM
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ITC: enzyme kinetics with CPM
CPM
Bz-Ala-Arg
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ITC: enzyme kinetics with CPM
CPM
Bz-Ala-Arg
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ITC: enzyme kinetics with CPM
CPM
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
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CPM: comparison between methods
Substrate or
Inhibitor
Published
Dansyl-Ala-Arg Km = 0.083 mM kcat = 17 s-1
(fluorescence)
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PEAQ-ITC
Km = 0.183 mM kcat = 4.8 s-1 (MI)
Km = 0.210 mM kcat = 30.8 s-1 (SI)
Bz-Ala-Arg
Km = 0.162 mM kcat = 107 s-1 Km = 0.055 mM kcat = 136 s-1 (MI)
(HPLC)
Km = 0.484 mM kcat = 204 s-1 (MI)
Km = 0.701 mM kcat = 60 s-1 (SI)
Bradykinin
Km = 0.04 mM kcat = 2.1 s-1
(coupled enzyme assay)
Km = 0.071 mM kcat = 4.7 s-1 (MI)
Km = 0.053 mM kcat = 46 s-1 (SI)
MGTA
Ki = 2 – 3 nM
Ki = 1 – 10 nM
CPM: comparison between methods
Substrate or
Inhibitor
Published
Dansyl-Ala-Arg Km = 0.083 mM kcat = 17 s-1
(fluorescence)
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PEAQ-ITC
Km = 0.183 mM kcat = 4.8 s-1 (MI)
Km = 0.210 mM kcat = 30.8 s-1 (SI)
Bz-Ala-Arg
Km = 0.162 mM kcat = 107 s-1 Km = 0.055 mM kcat = 136 s-1 (MI)
(HPLC)
Km = 0.484 mM kcat = 204 s-1 (MI)
Km = 0.701 mM kcat = 60 s-1 (SI)
Bradykinin
Km = 0.04 mM kcat = 2.1 s-1
(coupled enzyme assay)
Km = 0.071 mM kcat = 4.7 s-1 (MI)
Km = 0.053 mM kcat = 46 s-1 (SI)
MGTA
Ki = 2 – 3 nM
Ki = 1 – 10 nM
CPM: trouble shooting
Only 50% active sites available
Why is Km affected?
Recycled enzyme needs QC
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Methods used for PRCP
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
PRCP
 HPLC: release of Phe from Z-Pro-Phe
 HPLC: release of Z-Pro from Z-Pro-Phe
 Mass spectrometry
C18 ZipTip
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ITC: enzyme kinetics with PRCP
DP (μW)
Multiple injections: raw calorimetric data
• 12 injections were made every 60 s
• short time gaps between injections to ensure
that steady-state conditions are maintained
and no more than 5% of the substrate is
depleted prior to the next injection
Time (min)
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Hydrolysis of angiotensin III by rPRCP
Multiple injections: Michaelis-Menten fitting via Orgin7 software
ΔH = -1.7 kcal/mol
ITC
Km = 0.40 ± 0.03 mM
kcat = 40.3 ± 1.2 s-1
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≅
RP-HPLC
Km = 0.33 ± 0.04 mM
kcat = 39.0 ± 1.9 s-1
Hydrolysis of (pyr)-apelin-13 by rPRCP
Multiple injections: raw calorimetric data
12 injections were made every 60 s
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Hydrolysis of (pyr)-apelin-13 by rPRCP
Multiple injections: Michaelis-Menten fitting via Orgin7 software
ΔH = -0.2 kcal/mol
ITC
Km = 0.50 ± 0.05 mM
kcat = 163 ± 6 s-1
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comparable to
angiotensin III
Method comparison PRCP
✔
-
RP-HPLC
Sensitive
Quantitative
analysis of
enzyme kinetics
- Time consuming
✗
-
-
-
(1 day)
High peptide &
enzyme
consumption
Optimization for
every substrate
(type AA released)
End-point
measurement
✔ MALDI-TOF
✔
-
-
Sensitive
Information on
cleavage site
- Time consuming
✗
-
-
-
(1-2 days)
Not compatible
for peptides with
low MW ( < 800)
Not optimal for
quantitative
analysis
End-point
measurement
-
-
ITC
Sensitive
Quantitative
analysis of enzyme
kinetics
Fast (1-2 hours)
Lower peptide
consumption
Compatible with
every substrate
Native conditions
- High enzyme
✗
-
consumption
Optimization of
buffer, enzyme
and substrate
concentrations
Conclusion
[email protected]
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The collaboration with Malvern was educational and
inspirational
ITC is a valuable alternative for kinetic analysis of
otherwise difficult enzymes
Amount of enzyme > other kinetic methods
Quality control of enzyme preparations
We aim to build on this experience to become a
reference lab for the use of ITC in enzyme kinetics