Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme.
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.
Reiss
Y
Y
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
Brown
M S
MS
Goldstein
J L
JL
D019883:Alkyl and Aryl Transferases; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008667:Metalloproteins / Q000378:metabolism* / Q000494:pharmacology; D008954:Models, Biological; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D051381:Rats; D012717:Sesquiterpenes; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D002413:Cations, Divalent; D008667:Metalloproteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D004492:Edetic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D008274:Magnesium; D015032:Zinc; C002963:geranylgeranyl pyrophosphate