Share Email Print
cover

Proceedings Paper

Microwave-optical study of an As(III) derivative of Eco RI methylase
Author(s): August H. Maki; Desiree H. H. Tsao
Format Member Price Non-Member Price
PDF $14.40 $18.00

Paper Abstract

The authors report on the formation of an unusually stable As(III)-thiolate with a single 'high- affinity' cysteine (Cys) of E. coli RI methylase, monitored via its influence on a neighboring tryptophan (Trp) residue in the enzyme structure. The binding was studied by Trp fluorescence quenching, low temperature phosphorescence and triplet state optically detected magnetic resonance (ODMR) of the intrinsic Trp residue(s). The affected Trp is subject to an external heavy atom effect (HAE) from arsenic, quenching its fluorescence and reducing its phosphorescence lifetime from 6 sec to ca. 70 msec. The enzyme high affinity binding site has at least 27 times the affinity for As(III) as does a typical sulfhydryl reagent, HSCH2CONH2. The accessibility of the arsenical to this Cys site was reduced upon formation of the ternary complex methylase-DNA-sinefungin, suggesting a local conformational change in the enzyme when DNA is bound. The enzymatic activity assay of methylase is not affected by the addition of a 1:1 molar ratio of the arsenical to the methylase, but incubation with an excess of As(III) causes complete loss of enzymatic activity. This suggests that the high- affinity Cys residue is not a part of the active site of the enzyme, but the addition of a molar excess of arsenical to the enzyme derivatizes the Cys residue known to be located in the active site.

Paper Details

Date Published: 1 June 1991
PDF: 10 pages
Proc. SPIE 1432, Biomolecular Spectroscopy II, (1 June 1991); doi: 10.1117/12.44212
Show Author Affiliations
August H. Maki, Univ. of California/Davis (United States)
Desiree H. H. Tsao, Univ. of California/Davis (United States)


Published in SPIE Proceedings Vol. 1432:
Biomolecular Spectroscopy II
Robert R. Birge; Laurence A. Nafie, Editor(s)

© SPIE. Terms of Use
Back to Top