The use of chromogenic reference substrates for the kinetic analysis of penicillin acylases

Wynand B.L. Alkema, René Floris, Dick B. Janssen

Onderzoeksoutput: ArticleAcademicpeer review

Samenvatting

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low Km values for these substrates, the lack of a spectroscopic signal, and the strong product inhibition by phenylacetic acid. To overcome these difficulties, a spectrophotometric method was developed, with which kinetic parameters could be determined by measuring the effects on the hydrolysis of the chromogenic reference substrate 2-nitro-5-[(phenylacetyl)amino]benzoic acid (NIPAB). To that end, spectrophotometric progress curves with NIPAB in the absence and presence of the phenylacetylated substrates and their products were measured and analyzed by numerical fitting to the appropriate equations for competing substrates with product inhibition. This analysis yielded kinetic constants for phenylacetylated substrates such as penicillin G, which are in close agreement with those obtained in independent initial velocity experiments. Using NIPAB analogs with lower kcat/Km values, kinetic parameters for the hydrolysis of cephalexin and penicillin V were determined. This method was suitable for determining the kinetic constants of penicillin acylases in periplasmic extracts from Escherichia coli, Alcaligenes faecalis, and Kluyvera citrophila. The use of chromogenic reference substrates thus appears to be a rapid and reliable method for determining kinetic constants with various substrates and enzymes.
Originele taal-2English
Pagina's (van-tot)47-53
Aantal pagina's7
TijdschriftAnalytical Biochemistry
Volume275
Nummer van het tijdschrift1
DOI's
StatusPublished - 1 nov. 1999
Extern gepubliceerdJa

Keywords

  • chromogene referentiesubstraten
  • kinetische analyse
  • penicilline-acylasen

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