The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-α-carbobenzoxyglycine p-nitrophenyl ester

Paolo Ascenzi, Patrizia Aducci, Antonio Torroni, Gino Amiconi, Alessandro Ballio, Enea Menegatti, Mario Guarneri

Research output: Contribution to journalArticlepeer-review

Abstract

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.2)-catalyzed hydrolysis of N-α-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 ± 0.5°C. The results are consistent with the minimum three-step mechanism involving the acyl · enzyme intermediate E·P: {A figure is presented} The formation of the E · S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 μM-1 s-1 (pH ≤ 3.5) and 0.40 μM-1 s-1 (pH > 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of K+2 Ks (= kcat Km; Ks = k-1 k+1) reflects the ionization of two groups with pK″ values of 4.5 ± 0.1 and 8.80 ± 0.15 in the free enzyme. The pH dependence of K+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK″ values of 5.80 - 0.15 and 3.10 - 0.15, respectively. As expected from the pH dependences of K+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 · 101 μM (pH ≤ 3.5) to 1.5 · 103 μM (pH > 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (≤3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.

Original languageEnglish
Pages (from-to)203-210
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume912
Issue number2
DOIs
Publication statusPublished - Apr 8 1987

Keywords

  • (C. papaya L.)
  • Cysteine proteinase
  • Papain
  • pH effect
  • Proteinase kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

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