The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

Abstract

Determination of kinetic parameters of penicillin acylases for phenylacetylated compounds is complicated due to the low K m 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 k cat /K m 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.

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…Although PA has intensively been studied due to this industrial importance (e.g. Alkema et al, 2000, Arroyo et al, 2003, Giordano et al, 2006, Kheirolomoom et al, 2001, published parameter estimates vary drastically (Alkema et al, 1999). To investigate if such deviations may be caused by using different analysis approaches, PA was chosen for the comparison of the computer programs.…”

“…Mixing was achieved by using a magnetic stir bar. The increase in absorbance at 405 nm due to the formed ANB (Alkema et al, 1999) was measured using an Uvikon 922 spectrophotometer (Kontron Instruments, Groß-Zimmern, Germany). In total nine experimental progress curves were measured (Table 2-1).…”

Self‐renaturing enzymes: Design of an enzyme‐chaperone chimera as a new approach to enzyme stabilization

“…Activity of recombinant PGA and soluble chimeras in aqueous buffer was determined by measuring the change in absorbance at 405 nm upon the addition of 90 mM 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB) (Alkema et al, 1999) to 10 mM enzyme in 50 mM Tris/HCl buffer (pH 7) containing 5 mM CaCl 2 . Reactions proceeded for 1 h and were run at least in triplicate.…”

Saturation mutagenesis reveals the importance of residues αR145 and αF146 of penicillin acylase in the synthesis of β-lactam antibiotics

“…Potassium phosphate buffer (1 M, pH 7.0) was added to the resulting periplasmic extract to a final concentration of 50 mM. The enzyme concentrations of the periplasmic extracts and pure enzymes of the ␣R145 mutants and mutant ␣F146H were determined using phenylmethylsulfonylfluoride (PMSF) titration (Svedas et al, 1977;Alkema et al, 1999). The conversion experiments were carried out at 30 • C in 50 mM potassium phosphate buffer, pH 7.0.…”