Kinetics of enzyme acylation and deacylation in the penicillin acylase-catalyzed synthesis of β-lactam antibiotics

Wynand B L Alkema, Erik de Vries, René Floris, Dick B Janssen

Research output: Contribution to journalArticleAcademicpeer-review


Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic β-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and D-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme. The phenylacetyl- and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s-1 and 75 s -1, respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the kcat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the kcat of NIPAOB resulted from an inverse isotope effect at low concentrations of D2O, which is overridden by a large normal isotope effect at large molar fractions of D2O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D 2O compared to H2O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The β-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steady-state, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of β-lactam antibiotics were obtained.
Original languageEnglish
Pages (from-to)3675-3683
Number of pages9
JournalEuropean Journal of Biochemistry
Issue number18
Publication statusPublished - Sept 2003
Externally publishedYes


  • acylation
  • anti-bacterial agents/biosynthesis
  • binding sites
  • binding, competitive
  • catalysis
  • cephalosporins/chemistry
  • deuterium
  • escherichia coli/enzymology
  • glycine/analogs & derivatives
  • hydrolysis
  • kinetics
  • penicillanic acid/analogs & derivatives
  • penicillin amidase/chemistry
  • phenylacetates/chemistry
  • protons
  • spectrophotometry, ultraviolet/methods
  • substrate specificity


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