Selenophosphate synthetase. Enzyme properties and catalytic reaction

• Published in: The Journal of biological chemistry Vol. 269; no. 14; pp. 10597 - 10603
• Main Authors: Veres, Z, Kim, I Y, Scholz, T D, Stadtman, T C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 08-04-1994
• Subjects: Adenine Nucleotides - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Catalysis; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Enzymes and enzyme inhibitors; Escherichia coli; Escherichia coli Proteins - antagonists & inhibitors; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Metals; Phosphotransferases - metabolism; Selenium - metabolism; Substrate Specificity; Transferases; Regulation(control); Enzymatic activity; Enzyme; Escherichia coli; Transferases; Reaction mechanism; Bacteria; Cations; Kinetics; Selenium; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)34101-7

Selenophosphate synthetase, the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate,
from ATP and selenide. Isolation of the enzyme and characterization of some of its physical and catalytic properties are described.
Magnesium ion and a monovalent cation, K+, NH4+, or Rb+, are required for catalytic activity. Polyphosphates and other common
nucleotide triphosphates do not replace ATP as substrate. The stoichiometry of the catalytic reaction (Reaction 1) was established
using 31P NMR, anaerobic molecular sieve chromatography, and radiochemical labeling procedures. ATP+selenide+H2O-->selenophosphate+Pi+AMP.
In the absence of selenide, ATP is converted completely to AMP and orthophosphate upon prolonged incubation with elevated
levels of enzyme. AMP is a competitive inhibitor of ATP, Ki = 170 microM, whereas selenophosphate and orthophosphate are weak
inhibitors indicating a multistep reaction. Attempts to obtain direct evidence for a postulated enzyme-pyrophosphate intermediate
using several experimental approaches are described. No exchange of [14C]AMP with ATP could be detected after the enzyme was
freed of traces of contaminating adenylate kinase by chromatography on phenyl-Sepharose.