Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

Martin Willemoës; Dan Nilsson; Bjarne Hove-Jensen

doi:10.1021/bi9528560

Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

Martin Willemoës, Dan Nilsson, Bjarne Hove-Jensen

15 Citations (Scopus)

Abstract

The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in K_M for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, or Cd²⁺, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co²⁺ and the D221A enzyme in the presence of Mn²⁺ or Co²⁺. The D220F and D221A enzymes both showed large decreases in V_app in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co²⁺. V_app of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg²⁺, Mn²⁺, or Cd²⁺, whereas the V_app was increased in the presence of Co²⁺. V_app values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg²⁺ or Mn²⁺, respectively, whereas V_app was similar to that of the wild-type and K_M for Rib-5-P was increased 4-fold in the presence of Cd²⁺. The changes in K_M for ribose 5-phosphate and V_app of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis.

Original language	English
Journal	Biochemistry
Volume	35
Issue number	25
Pages (from-to)	8181-8186
ISSN	0006-2960
DOIs	https://doi.org/10.1021/bi9528560
Publication status	Published - 1996

Access to Document

10.1021/bi9528560

Fingerprint

Dive into the research topics of 'Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding'. Together they form a unique fingerprint.

Cite this

Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding. / Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne.

In: Biochemistry, Vol. 35, No. 25, 1996, p. 8181-8186.

Research output: Contribution to journal › Journal article › Research › peer-review

@article{d0136640813e11dcbee902004c4f4f50,

title = "Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding",

abstract = "The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, whereas Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+. The changes in KM for ribose 5-phosphate and Vapp of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis. ",

author = "Martin Willemo{\"e}s and Dan Nilsson and Bjarne Hove-Jensen",

year = "1996",

doi = "10.1021/bi9528560",

language = "English",

volume = "35",

pages = "8181--8186",

journal = "Biochemistry",

issn = "0006-2960",

publisher = "American Chemical Society",

number = "25",

}

TY - JOUR

T1 - Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

AU - Willemoës, Martin

AU - Nilsson, Dan

AU - Hove-Jensen, Bjarne

PY - 1996

Y1 - 1996

N2 - The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, whereas Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+. The changes in KM for ribose 5-phosphate and Vapp of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis.

AB - The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, whereas Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+. The changes in KM for ribose 5-phosphate and Vapp of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis.

U2 - 10.1021/bi9528560

DO - 10.1021/bi9528560

M3 - Journal article

C2 - 8679571

SN - 0006-2960

VL - 35

SP - 8181

EP - 8186

JO - Biochemistry

JF - Biochemistry

IS - 25

ER -