Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Journal Article · Thu Mar 15 04:00:00 EST 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6968199

Som, S; Friedman, S ^[1]

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1. State Univ. of New York Health Science Center, Brooklyn (USA)

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either (methyl-3H)AdoMet or (35S)AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of (3H)AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported. The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.

• OSTI ID: 6968199
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:8; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560130* -- Radiation Effects on Microorganisms
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ADDUCTS
ADENOSINE
AFFINITY
AMINO ACIDS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CHEMICAL RADIATION EFFECTS
CHEMICAL REACTIONS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DNA ADDUCTS
DRUGS
ELECTROMAGNETIC RADIATION
ELECTROPHORESIS
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MEMBRANE PROTEINS
METHIONINE
METHYL TRANSFERASES
METHYLATION
MICROORGANISMS
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOTOCHEMISTRY
PROTEINS
RADIATION CHEMISTRY
RADIATION EFFECTS
RADIATIONS
RADIOINDUCTION
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RIBOSIDES
SUBSTRATES
SULFUR 35
SULFUR ISOTOPES
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION