Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme

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Abstract

The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.

Original languageEnglish (US)
Pages (from-to)6403-6408
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number9
StatePublished - Mar 25 1992

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Divalent Cations
Zinc
Rats
Brain
Binding Sites
Enzymes
Carboxypeptidases A
Dialysis
Metalloproteases
Enzyme Inhibitors
Peptidyl-Dipeptidase A
Substrates
Edetic Acid
Leucine
Gel Chromatography
Gels
Peptides
diphosphoric acid
prenyl
p21(ras) farnesyl-protein transferase

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme",
abstract = "The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.",
author = "Yuval Reiss and Brown, {Michael S.} and Goldstein, {Joseph L.}",
year = "1992",
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T1 - Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme

AU - Reiss, Yuval

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AU - Goldstein, Joseph L.

PY - 1992/3/25

Y1 - 1992/3/25

N2 - The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.

AB - The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.

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