Involvement of the alpha subunit of farnesyl-protein transferase in substrate recognition.
Using photoaffinity labeling, we have identified a region in mammalian farnesyl-protein transferase (FPTase) involved in substrate recognition. The photolabel used (Compound 1) is a peptide containing the photoactive amino acid p-benzoylphenylalanine (Bpa). Upon exposure to UV light. Compound 1 inhibits FPTase activity in a time- and concentration-dependent manner. Photoinhibition of FPTase activity by Compound 1 is prevented by adding H-Ras to the reaction mixture, indicating that labeling is targeted to the enzyme active site. We used peptide mapping by HPLC, Edman sequencing, and matrix-assisted time-of-flight (MALDI-TOF) mass spectrometry to identify the site of interaction with radiolabeled Compound 1. These experiments indicate that a specific region of the alpha subunit of the enzyme, Asp110-Arg112, is involved in substrate binding and suggest that Glu111 is likely to be the residue covalently modified by the photoaffinity label. Sequence alignments between yeast and mammalian FPTases reveal that Glu111 is conserved. The implications of this finding are discussed in light of previous mutagenesis studies on FPTase.
Pellicena
P
P
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook 11794, USA.
Scholten
J D
JD
Zimmerman
K
K
Creswell
M
M
Huang
C C
CC
Miller
W T
WT
D000345:Affinity Labels; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004151:Dipeptides; D006801:Humans; D008969:Molecular Sequence Data; D010777:Photochemistry; D010782:Photolysis; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000345:Affinity Labels; D004151:Dipeptides; D011994:Recombinant Proteins; C101937:benzyloxycarbonyl-histidyl-N-(2-(phenylmethoxy)ethyl)(4-benzoyl)phenylalaninamide; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase