Photoreactivating Enzyme from Escherichia coli: Isolated Enzyme Lacks Absorption in Its Actinic Wavelength Region and Its Ribonucleic Acid Cofactor Is Partially Double Stranded When Associated with Apoprotein

George D. Cimino, John C. Sutherland

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 °C and subsequently cooling to 4 °C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (µ= 0.01), heating to 15 °C or protease treatment at 4 °C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 °C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.

Original languageEnglish (US)
Pages (from-to)3914-3921
Number of pages8
JournalBiochemistry
Volume21
Issue number17
DOIs
StatePublished - Jan 1 1982
Externally publishedYes

Fingerprint

Deoxyribodipyrimidine Photo-Lyase
Apoproteins
Escherichia coli
Base Pairing
RNA
Wavelength
Enzymes
Heating
Polynucleotides
Denaturation
Density (optical)
Circular Dichroism
Ionic strength
Light scattering
Osmolar Concentration
Conformations
Absorption spectra
Peptide Hydrolases
Hot Temperature
Association reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{b5aafeaca2bc43e1b24704c1042d9afc,
title = "Photoreactivating Enzyme from Escherichia coli: Isolated Enzyme Lacks Absorption in Its Actinic Wavelength Region and Its Ribonucleic Acid Cofactor Is Partially Double Stranded When Associated with Apoprotein",
abstract = "Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 °C and subsequently cooling to 4 °C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (µ= 0.01), heating to 15 °C or protease treatment at 4 °C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 °C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20{\%} are in a double-stranded conformation in the native enzyme.",
author = "Cimino, {George D.} and Sutherland, {John C.}",
year = "1982",
month = "1",
day = "1",
doi = "10.1021/bi00260a002",
language = "English (US)",
volume = "21",
pages = "3914--3921",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "17",

}

TY - JOUR

T1 - Photoreactivating Enzyme from Escherichia coli

T2 - Isolated Enzyme Lacks Absorption in Its Actinic Wavelength Region and Its Ribonucleic Acid Cofactor Is Partially Double Stranded When Associated with Apoprotein

AU - Cimino, George D.

AU - Sutherland, John C.

PY - 1982/1/1

Y1 - 1982/1/1

N2 - Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 °C and subsequently cooling to 4 °C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (µ= 0.01), heating to 15 °C or protease treatment at 4 °C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 °C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.

AB - Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60 °C and subsequently cooling to 4 °C release the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (µ= 0.01), heating to 15 °C or protease treatment at 4 °C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4 °C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairs are present in equal concentrations and that about 20% are in a double-stranded conformation in the native enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0020410589&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020410589&partnerID=8YFLogxK

U2 - 10.1021/bi00260a002

DO - 10.1021/bi00260a002

M3 - Article

C2 - 6181804

AN - SCOPUS:0020410589

VL - 21

SP - 3914

EP - 3921

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 17

ER -