PROLINE SPECIFIC ENDOPEPTIDASE from Flavobacterium sp.

PSP-101

Prolyl endopeptidase (EC 3.4.21.26)
X-Prolyl-peptide +H₂O                      ► X-Proline + Peptide

PREPARATION and SPECIFICATION
Appearance:  White amorphous powder, lyophilized
Activity:  GradeⅠ 5.0U/mg-solid or more
Contaminants:  Leucine aminopeptidase ≤1.0×10⁻¹%
Trypsin-like activity ≤1.0×10⁻¹%
PROPERTIES
Stability:  Stable at -20°C for at least One year (Fig.1)
Molecular weight :  approx. 78,000
Isoelectric point:  9.1
Michaelis constants:  2.5×10⁻⁵M (Z-Gly-Pro-MCA)
1.4×10⁻⁴M (Z-Gly-Pro-2NNap)
Structure:  Monomer
Inhibitors:  DFP, 3, 4-dichloroisocoumarin, Z-Gly-Pro-CH₂Cl
Optimum pH :  6.5(Fig.2)
Optimum temperature :  37°C (40°C) ² ⁾(Fig.3)
pH Stability :  5.5-8.5 (30°C,15hr)(Fig.4)
Thermal stability :  below 40°C (pH7.0,10min)(Fig.5)
Substrate specificity:  Y-Pro(Ala)-X (Y, peptide or N-protected amino acid; X, amino acid,
peptide, amide, or ester)(Table 1)
Effect of various chemicals :  (Table 2)

APPLICATIONS

This enzyme is useful for the determination of amino acid sequences of peptides and proteins containing proline residues.

ASSAY

Principle:

proline specific endopeptidase

Carbobenzoxy-Gly-Pro-p-nitroanilide(Z-Gly-Pro-pNA)+H₂O                                                  ►
Z-Gly-Pro+p-Nitroaniline

The appearance of p-nitroaniline is measured at 410nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of p-nitroaniline per minute under the conditions described below.

Method:

Reagents
A. K-Phosphate buffer, pH 7.0:  0.1M
B. Z-Gly-Pro-pNA solution:  5mM[Dissolve 21.3mg of Z-Gly-Pro-pNA(MW=426.43) in ca.8ml of 40%
dioxane in a hot bath at 60°C, then cool down to 25°C, fill up to 10ml with 40% dioxane](Should be prepared fresh)
C. Acetate buffer, pH 4.0:  1M solution containing 10% of TritonX-100 (Store at 5°C)
D. Enzyme diluent:  50mM K-phosphate buffer, pH 7.0

Procedure

Concentration in assay mixture
Phosphate buffer 77.8 mM
Z-Gly-Pro-pNA 0.926 mM

1. Prepare the following reaction mixture in a test tube, and equilibrate at 30°C for about 5 minutes.

1.0 ml K-Phosphate buffer (A)
0.25 ml Substrate solution (B)

2. Add 0.1ml of enzyme solution* and mix by gentle inversion.

3. After exactly 5 minutes at 30°C, add 2.0ml of acetate buffer (C) to stop the reaction and measure the optical
density at 410nm against water (OD test).
At the same time, prepare the blank by first mixing the reaction mixture with 2.0ml of acetate buffer (C) after 5min-incubation at 30°C, followed by the addition of the enzyme solution (OD blank).

* Immediately before assay,dissolve the enzyme preparation in ice-cold enzyme diluent (D) and dilute to
0.05-0.2U/ml with the same buffer.

Calculation

Activity can be calculated by using the following formula :

ΔOD (OD test-OD blank)×Vt×df

Volume activity (U/ml) =                                                               = ΔOD×1.20×df

5.57×1.0× t ×Vs


Weight activity (U/mg)=(U/ml)×1/C

Vt
: Total volume (3.35ml)
Vs
: Sample volume (0.1ml)
5.57
: Millimolar extinction coefficient of p-nitroaniline under the assay condition (㎠/micromole)
1.0
: Light path length (cm)
t
: Reaction time (5 minutes)
df
: Dilution factor
 
C
: Enzyme concentration in dissolution (c mg/ml)
 

REFERENCES

  1. T.Yoshimoto and D.Tsuru; Agric.Biol.Chem., 42, 2417 (1978)
  2. T.Yoshimoto, R.Walter and D.Tsuru; J.Biol.Chem., 255, 4786(1980)
  3. R.Walter; Biochim.Biophys.Acta.,422, 132 (1976)
  4. T.Yoshimoto, R.C.Olowski and R.Walter; Biochem.J., 16, 2942 (1977)
  5. T.Yoshimoto, M.Fischl, R.C.Olowski and R.Walter; J.Biol.Chem., 253, 3708 (1978)
  6. T.Yoshimoto and D.Tsuru; Protein,Nucleic acid and Enzyme (Japanese), 29, 127 (1984)
  7. L.Haeffiner-Gormley, L.Parente and D.B.Wetlaufer; Int.J.Peptide Protein Res., 26, 83 (1985)
  8. T.Yoshimoto, K.Kawahara, F.Matsubara, K.Kado and D.Tsuru; J.Biochem., 98, 975 (1985)
Table 1. Substrate Specificity of Proline specific endopeptidase
Substrate Km
(mM)		 kcat
(s⁻¹)		 kcat/Km
(mM⁻¹・S⁻¹)
↓      
Pro-2NNap   not hydrolyzed  
Z-Pro-2NNap
not hydrolyzed  
Gly-Pro-2NNap   not hydrolyzed  
Z-Gly-Pro-MCA 0.025 115 4600
Z-Gly-Pro-2NNap 0.14 169 1212
Z-Gly-Pro-pNP 0.125 102 816
Z-Ala-Pro-2NNap 0.08 642 834
Z - D -Pro-2NNap 0.20 0.142 0.73
Ala-      
Z-Ala-Gly-Pro-2NNap 0.29 192 664
Z-D-Ala-Gly-Pro-2NNap 0.14 38 271
Z-Gly-Pro-Leu 0.22 23 104
Z-Gly-Pro-Phe 0.74 180 250
Z-Gly-Pro-ALa 0.39 240 620
Z-Gly-Pro-D-Ala   not hydrolyzed  
Z-Gly-Pro-Leu-Gly 0.32 520 1600
Z-Gly-Pro-Leu-Ala 0.42 520 1100
Z-Gly-Pro-Leu-D-Ala 1.5 1600 1070
Z-Gly-Pro-Leu-Gly-Gly 1.4 700 500
Z-Gly-Pro-Leu-Gly-Ala 1.82 1000 550

Z, Carbobenzoxy; MCA, 4-Methyl-coumaryl-7-amide; 2NNap, ß-Naphthylamide; pNP, p-Nitrophenyl ester.

Table 2. Effect of Various Chemicals on Proline specific endopeptidase
[The enzyme dissolved in 50mM K-phosphate buffer, pH 7.0 (2.5U/ml) was incubated with each chemical at 25 °C for 30min.]
Chemical Concn.(mM) Residual
activity(%)		 Chemical Concn.(mM) Residual
activity(%)
None − 100 NaF 2.0 76.9
Metal salt 2.0   NaN₃ 20 77.3
MgCl₂   72.1 DFP 1.0 2.7
CaCl₂   74.6 o-Phenanthroline
2.0 84.9
BaCl₂   74.3
α,α′-Dipyridyl 1.0 84.9
FeCl₃   52.6 Borate 50 73.4
CoCl₂   62.7 IAA 2.0 62.8
MnCl₂   68.9
NEM 2.0 74.4
ZnSO₄   56.8 Hydroxylamine 2.0 77.4
Cd(OAc)₂   31.9 3,4-Dichloroiso-coumarin 2.0 9.0
NiCl₂   66.8 Triton X-100 0.10% 86.4
CuSO₄   45.2 Brij 35 0.10% 84.8
Pb(OAc)₂   59.8
Tween 20
0.10% 84.1
AgNO₃   55.7 Span 20 0.10% 81.9
HgCl₂   0 Na-cholate 0.10% 84.1
PCMB 2.0 71.9 SDS 0.05% 73.0
MIA 2.0 78.1
      

Ac, CH₃CO; PCMB, p-Chloromercuribenzoate; MIA, Monoiodoacetate; DFP, Diisopropylphosphorofluoridate; IAA, Iodoacetamide; NEM, N-Ethylmaleimide; SDS, Sodium dodecyl sulfate.

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