Colorimetric assay of penicillin amidase activity using phenylacetyl-aminobenzoic acid as substrate

Szewczuk, A; Siewiński, Maciej; Słowińska, R

doi:10.1016/0003-2697(80)90251-1

Cited by 22 publications

(12 citation statements)

References 8 publications

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“…The values obtained from progress curves for the hydrolysis of NIPAB were in good agreement with the values obtained with the initial rate experiments and with data reported by Kutzbach and Rauenbusch (8) and Kasche et al (14). For phenylacetic acid, inhibition constants ranging from 0.05 to 5 mM have been reported (3,7,8,10). However, the K m and K i values for other phenylacetylated compounds are all in the range of 10 to 200 M (9,14), which indicates that the K i value of 70 M for phenylacetic acid obtained in these experiments is correct.…”

Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 68%

“…When both substrates are present in the reaction mixture, the relative rates at which the chromogenic substrate (V chrom ) and the invisible substrate (V invis ) are converted are given by Eq. [7] (11):…”

Section: New Chromogenic Substratesmentioning

confidence: 99%

“…Moreover, the classical steady-state method of measuring initial rates is prone to severe error because of the strong product inhibition by phenylacetic acid. Due to these complications, K m values for penicillin G and K i values for phenylacetic acid of penicillin acylase of E. coli have been reported, which differ by several orders of magnitude (3,(7)(8)(9)(10).…”

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confidence: 99%

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

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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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.

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Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 68%

Section: New Chromogenic Substratesmentioning

confidence: 99%

mentioning

confidence: 99%

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

Alkema

Floris

Janssen

1999

Analytical Biochemistry

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“…The K m was 7.5 Ϯ 0.9 M compared to 3.8 M for benzylpenicillin (33). Hence, the affinity of E. coli penicillin G acylase for phenylacetyl-MCA was found to be 3.5 times higher than for phenylacetyl-p-nitro-m-carboxyanilide (16), 4 times higher than for NIPAB (33), and 24 times higher than for phenylacetyl-4-aminobenzoic acid (15). Not surprisingly, the novel substrate allows a rather sensitive detection of penicillin G acylase activity.…”

Section: Resultsmentioning

confidence: 92%

Fluorogenic Assay for Penicillin G Acylase Activity

Ninkovic¹,

Riester²,

Wirsching³

et al. 2001

Analytical Biochemistry

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“…The construction of the PA mutants was done using the protocols of the Muta-Gene in vitro mutagenesis kit of Bio-Rad Laboratories (Bio-Rad 1987), Phenylacetyl-4-aminobenzoic acid (PAPABA) was prepared as described by Szewczuk et al (1980). Pen G and 6-APA were supplied by Antibirticos (Leon, Spain) Penicillin V (Pen V), fluorescamine and p-dimethylaminobenzaldehyde were purchased from Sigma (St. Louis, Mo, USA) Phenylmethylsulphonyl fluoride (PMSF) and other analytical grade reagents were from Merck (Darmstadt, FRG).…”

Section: Methodsmentioning

confidence: 99%

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

Priéto¹,

Martín

Arche

et al. 1990

Appl Microbiol Biotechnol

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Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant kcat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the kcat and phenylmethylsulphonyl fluoride reactivity, but change the Km. The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.

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Colorimetric assay of penicillin amidase activity using phenylacetyl-aminobenzoic acid as substrate

Cited by 22 publications

References 8 publications

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

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

Fluorogenic Assay for Penicillin G Acylase Activity

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

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