Comparison of several new chromogenic galactosides as substrates for various β-d-galactosidases

I. Pócsi, S. A. Taylor, A. C. Richardson, B. V. Smith, R. G. Price

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

The kinetic characteristics of β-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli β-d-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed interactions not involving the catalytic site. When the product of the maximum observed velocity (vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The β-d-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.

Original languageEnglish
Pages (from-to)54-60
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1163
Issue number1
DOIs
Publication statusPublished - Apr 21 1993

Fingerprint

Galactosidases
Chromogenics
Galactosides
Substrates
Escherichia coli
Aspergillus niger
Enzyme Assays
Biotechnology
Substrate Specificity
Testis
Molecular Biology
Catalytic Domain
Hydrolysis
Oxygen
Liver
Assays
Enzymes
Molecular biology
Jacks
Aspergillus

Keywords

  • (A. niger)
  • (Bovine)
  • (E. coli)
  • (Jack bean)
  • Chromogenic substrate
  • β-d-Galactosidase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

Cite this

Comparison of several new chromogenic galactosides as substrates for various β-d-galactosidases. / Pócsi, I.; Taylor, S. A.; Richardson, A. C.; Smith, B. V.; Price, R. G.

In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1163, No. 1, 21.04.1993, p. 54-60.

Research output: Contribution to journalArticle

@article{849ad28d6c1a43b5903a4b734ead143c,
title = "Comparison of several new chromogenic galactosides as substrates for various β-d-galactosidases",
abstract = "The kinetic characteristics of β-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli β-d-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed interactions not involving the catalytic site. When the product of the maximum observed velocity (vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The β-d-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.",
keywords = "(A. niger), (Bovine), (E. coli), (Jack bean), Chromogenic substrate, β-d-Galactosidase",
author = "I. P{\'o}csi and Taylor, {S. A.} and Richardson, {A. C.} and Smith, {B. V.} and Price, {R. G.}",
year = "1993",
month = "4",
day = "21",
doi = "10.1016/0167-4838(93)90278-Y",
language = "English",
volume = "1163",
pages = "54--60",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Comparison of several new chromogenic galactosides as substrates for various β-d-galactosidases

AU - Pócsi, I.

AU - Taylor, S. A.

AU - Richardson, A. C.

AU - Smith, B. V.

AU - Price, R. G.

PY - 1993/4/21

Y1 - 1993/4/21

N2 - The kinetic characteristics of β-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli β-d-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed interactions not involving the catalytic site. When the product of the maximum observed velocity (vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The β-d-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.

AB - The kinetic characteristics of β-galactosidases from bovine liver and testes, Escherichia coli, Aspergillus niger and Jack bean were studied using five newly-developed colorimetric substrates. All the chromophores released by enzyme hydrolysis had high extinction coefficients in the visible region of the spectrum. Varying amounts of substrate inhibition were found with each of these substrates (VBzTM-Gal, VLM-Gal, VLPr-Gal, VQM-Gal and VQPr-Gal), but this was not a significant problem if the correct assay conditions were used. The substrates attached particularly tightly to the active centre of E. coli β-d-galactosidase resulting in low Km values. The data suggest that the chemical properties of the heterocyclic portion of the aglycone distant from the glycosidic oxygen do not affect the substrate specificity and the substrate inhibition can be attributed interactions not involving the catalytic site. When the product of the maximum observed velocity (vm) and the molar absorption coefficient is calculated for each substrate, the relative merits of the substrates for the assay of each enzyme can be assessed. The β-d-galactosidases from fungal and bacterial sources hydrolysed the substrates most efficiently, indicating that they may be of particular value in areas of molecular biology and biotechnology.

KW - (A. niger)

KW - (Bovine)

KW - (E. coli)

KW - (Jack bean)

KW - Chromogenic substrate

KW - β-d-Galactosidase

UR - http://www.scopus.com/inward/record.url?scp=0027478127&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027478127&partnerID=8YFLogxK

U2 - 10.1016/0167-4838(93)90278-Y

DO - 10.1016/0167-4838(93)90278-Y

M3 - Article

VL - 1163

SP - 54

EP - 60

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 1

ER -