Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenografT

Saeed Radparvar, Peter J Houghton, Janet A. Houghton

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6R]CH2,-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 μM) were >200-fold and [6R]CH2-H4PteGlu2 (18.2 μm) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 μM) at stabilizing ternary complexes for a T 1 2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 × 10-8 M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 × 10-9 M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 × 106M-1 min-1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Original languageEnglish (US)
Pages (from-to)335-342
Number of pages8
JournalBiochemical Pharmacology
Volume38
Issue number2
DOIs
StatePublished - Jan 15 1989
Externally publishedYes

Fingerprint

Fluorodeoxyuridylate
Thymidylate Synthase
Heterografts
Polyglutamic Acid
Colon
Adenocarcinoma
Folic Acid
5,10-methylenetetrahydrofolic acid
Tumors
Modulation
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

@article{416cab1be9114c58bdfd4c1aa7c0beec,
title = "Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenografT",
abstract = "CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6R]CH2,-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 μM) were >200-fold and [6R]CH2-H4PteGlu2 (18.2 μm) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 μM) at stabilizing ternary complexes for a T 1 2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 × 10-8 M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 × 10-9 M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 × 106M-1 min-1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.",
author = "Saeed Radparvar and Houghton, {Peter J} and Houghton, {Janet A.}",
year = "1989",
month = "1",
day = "15",
doi = "10.1016/0006-2952(89)90046-4",
language = "English (US)",
volume = "38",
pages = "335--342",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "2",

}

TY - JOUR

T1 - Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenografT

AU - Radparvar, Saeed

AU - Houghton, Peter J

AU - Houghton, Janet A.

PY - 1989/1/15

Y1 - 1989/1/15

N2 - CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6R]CH2,-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 μM) were >200-fold and [6R]CH2-H4PteGlu2 (18.2 μm) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 μM) at stabilizing ternary complexes for a T 1 2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 × 10-8 M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 × 10-9 M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 × 106M-1 min-1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

AB - CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6R]CH2,-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 μM) were >200-fold and [6R]CH2-H4PteGlu2 (18.2 μm) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 μM) at stabilizing ternary complexes for a T 1 2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 × 10-8 M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 × 10-9 M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 × 106M-1 min-1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

UR - http://www.scopus.com/inward/record.url?scp=0024578676&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024578676&partnerID=8YFLogxK

U2 - 10.1016/0006-2952(89)90046-4

DO - 10.1016/0006-2952(89)90046-4

M3 - Article

C2 - 2914018

AN - SCOPUS:0024578676

VL - 38

SP - 335

EP - 342

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 2

ER -