Substrate specificity of penicillin amidase from E. coli

Margolin, Alexey L.; Švedas, Vytas K.; Березин, И.В.

doi:10.1016/0005-2744(80)90145-x

Cited by 93 publications

(46 citation statements)

References 13 publications

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“…The kinetic values obtained with the E. coli penicillin acylase for the hydrolysis of NIPAB and penicillin G agree well with values obtained in literature (9,14). The k cat value of 170 s Ϫ1 reported for the hydrolysis of phenylacetic acid ethyl ester (9) is comparable to the value of 190 s Ϫ1 that was obtained for phenylacetic acid methyl ester and indicates that phenylacetylated esters are good substrates for penicillin acylase.…”

Section: Discussionsupporting

confidence: 89%

“…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. These results show that accurate kinetic constants were obtained by analysis of progress curves (Table 1).…”

Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 68%

“…(18) and (19). (9). The preferential conversion of cephalexin in the presence of 86 M NIPGB caused a typical S-shaped progress curve, which was not observed when 100 M NIPAB was the reference substrate, since only a small amount of the cephalexin was hydrolyzed under the latter conditions (Fig.…”

Section: New Chromogenic Substratesmentioning

confidence: 94%

“…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: Discussionsupporting

confidence: 89%

Section: Kinetics Of Nipab Hydrolysis and Phenylacetic Acid Inhibitionmentioning

confidence: 68%

Section: New Chromogenic Substratesmentioning

confidence: 94%

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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“…[4] The enzymatic synthesis of a b-lactam antibiotic such as ampicillin is not trivial, since a simple condensation of d-phenylglycine (d-PG) and 6-APA is thermodynamically unfavourable. [5,6] In consequence, all viable synthesis schemes involve an activated side-chain donor, such as d-phenylglycine amide (d-PGA), which acylates 6-APA in the presence of penicillin acylase in a kinetically controlled reaction; [7] water is the reaction medium of choice. A major problem of such schemes is the competing irreversible hydrolysis of the acyl donor as well as the product, leading to unreactive d-PG (see Figure 1).…”

Section: Introductionmentioning

confidence: 99%

Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents

Langen¹,

Oosthoek²,

Rantwijk³

et al. 2003

Adv Synth Catal

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Penicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis, immobilised as a cross-linked enzyme aggregate (CLEA), catalysed the synthesis of ampicillin in water-miscible organic solvents at low water concentrations. Below 4% water (v/v) no reaction was observed, showing the crucial role of water in maintaining the activity of penicillin acylase. The initial value of S/H was strongly affected by the nature of the solvent, but the effect of the water content was slight in the studied range of 4 to 15%. A reaction in acetonitrile containing 8% water afforded ampicillin in up to 86% yield.

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