TY - JOUR
T1 - Biochemical analysis of Escherichia coli selenophosphate synthetase mutants. Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization
AU - Ick Young Kim, Young Kim
AU - Veres, Z.
AU - Stadtman, T. C.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1993
Y1 - 1993
N2 - A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme (Kim, I. Y., Veres, Z., and Stadtman, T. C. (1992) J. Biol. Chem. 267, 19650-19654) is located in a glycine-rich segment near the N terminus of the protein. The possibility that this peptide sequence (HGAGCGCK) defines the ATP-binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis. Thus His-13 and Gly- 18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln. Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln. The mutation of Cys-19 and His-13 did not substantially alter the ATP K(m) and V(max) values, whereas the Gly-18 mutation resulted in a 4-fold increase in the ATP K(m) value compared with that of the wild type. ATP binding properties of the mutant enzymes were determined using Mn-[32P]ATP or Mn-[14C]ATP and gel filtration. Photoaffinity labeling of the proteins with [γ-32P]8-azido-ATP showed that all mutant enzymes could be labeled with the ATP analog except those in which Cys-17 or Cys-19 were replaced with serine.
AB - A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme (Kim, I. Y., Veres, Z., and Stadtman, T. C. (1992) J. Biol. Chem. 267, 19650-19654) is located in a glycine-rich segment near the N terminus of the protein. The possibility that this peptide sequence (HGAGCGCK) defines the ATP-binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis. Thus His-13 and Gly- 18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln. Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln. The mutation of Cys-19 and His-13 did not substantially alter the ATP K(m) and V(max) values, whereas the Gly-18 mutation resulted in a 4-fold increase in the ATP K(m) value compared with that of the wild type. ATP binding properties of the mutant enzymes were determined using Mn-[32P]ATP or Mn-[14C]ATP and gel filtration. Photoaffinity labeling of the proteins with [γ-32P]8-azido-ATP showed that all mutant enzymes could be labeled with the ATP analog except those in which Cys-17 or Cys-19 were replaced with serine.
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M3 - Article
C2 - 8262938
AN - SCOPUS:0001172445
SN - 0021-9258
VL - 268
SP - 27020
EP - 27025
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 36
ER -