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Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Summary

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism:

$${\text{E + S}}\mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 1}}} } }\limits^{{\text{k}}_{{\text{ + 1}}} } {\text{E}} \cdot {\text{S}}\mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 2}}} } }\limits^{{\text{k}}_{{\text{ + 2}}} } {\text{E}} \cdot {\text{P + P}}_{\text{1}} \mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 3}}} } }\limits^{{\text{k}}_{{\text{ + 3}}} } {\text{E + P}}_{\text{2}} .$$

Under pre-steady-state conditions, where [E0] ≫ [S0], the values of the dissociation constant of the E · S complex (Ks = k−1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] ≫ [E0], the values of kcat and Km have been obtained over the same pH range.

The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 ± 0.10.

The values of Ks and Km are pH independent.

The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenylesters of N-α-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.β-trypsin and b.α-chymotrypsin.

For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0.

The different catalytic behaviours of p. p. elastase, b.β-trypsin and b.α-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.

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Abbreviations

p.p.:

porcine pancreatic

b.:

bovine

ZGlyONp:

N-α-carbobenzoxy-glycine p-nitrophenyl ester

ZAlaONp:

N-α-carbobenzoxy-L-alanine p-nitrophenyl ester

BocAlaONp:

N-α-terbutyloxycarbonyl-L-alanine p-nitrophenyl ester

BzAlaOMe:

N-α-benzoyl-L-alanine methyl ester

FaAlaOMe:

N-α-furyl-acryloyl-L-alanine methyl ester

ZlleONp:

N-α-carbobenzoxy-L-isoleucine p-nitrophenyl ester

ZLeuONp:

N-α-carbobenzoxy-L-leucine p-nitrophenyl ester

ZValONp:

N-α-carbobenzoxy-L-valine p-nitrophenyl ester

ZArgONp:

N-α-carbobenzoxy-L-arginine pnitrophenyl ester

ZLysONp:

N-α-carbobenzoxy-L-lysine p-nitrophenyl ester

ZTyrONp:

N-α-carbobenzoxy-L-tyrosine p-nitrophenyl ester

ZAla:

N-α-carbobenzoxy-L-alanine

pNTA:

p-nitrophenyl-trymethyl acetate

diEpNP:

diethyl-pnitrophenyl phosphate

NaDodSO4 :

sodium dodecyl sulfate

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Ascenzia, P., Menegatti, E., Guarneri, M. et al. Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study. Mol Cell Biochem 56, 33–38 (1983). https://doi.org/10.1007/BF00228766

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Keywords

  • Trypsin
  • Serine Protease
  • Catalytic Property
  • Dissociation Constant
  • Individual Rate