Catalytic properties of Ancrod, the thrombin-like proteinase from the Malayan pit viper (Agkistrodon rhodostoma) venom

Paolo Ascenzi, Alberto Bertollini, Martino Bolognesi, Mario Guarneri, Enea Menegatti, Gino Amiconi

Research output: Contribution to journalArticlepeer-review

Abstract

Kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy-l-amino acids catalyzed by Ancrod were determined between pH 5 and 10 (I = 0.1 M) at 21 ± 0.5°C; the results are consistent with the minimum three-step mechanism: E ⇋ k-1 k+1E·S → k+2 E·PP→E+P2.For all substrates examined, the pH profiles of Kcat and/or kcat/Km reflect the ionization of two groups with pKa values ranging between 6.9 and 7.2, and 9.3 and 9.6 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); at variance, values of Km are pH-independent. Moreover, the formation of the E · S complexes may be regarded as a pseudo-equilibrium process, and the acylation step (k+2) is always rate-limiting in catalysis. Among p-nitrophenyl esters examined, ZArgONp shows the most favourable kinetic parameters and may be the substrate of choice for Ancrod, in that it allows the determination of the enzyme concentration as low as 1 · 10-9) M ({reversed tilde equals} 0.1 Ancrod units/ ml), at the optimum pH value ({reversed tilde equals} 8). The catalytic behaviour of Ancrod is compared to that of serine proteinases acting on cationic and non-cationic substrates; differences in kinetics, which refer to a lower enzyme:substrate affinity, may be related to a higher rigidity, lower hydrophobicity and/or adverse steric hindrance of the S1 subsite of Ancrod.

Original languageEnglish
Pages (from-to)415-423
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume829
Issue number3
DOIs
Publication statusPublished - Jul 1 1985

Keywords

  • (A. rhodostoma)
  • pH effect
  • Proteinase kinetics
  • Snake venom
  • Synthetic substrate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

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