Involvement of the Subunit of Farnesyl-Protein Transferase in Substrate Recognition

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• 作者：Patricia Pellicena ; Jeffrey D. Scholten ; Karen Zimmerman ; Mark Creswell ; C. C. Huang ; and W. Todd Miller
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：October 15, 1996
• 年：1996
• 卷：35
• 期：41
• 页码：13494 - 13500
• 全文大小：331K
• 年卷期：v.35,no.41(October 15, 1996)
• ISSN：1520-4995

文摘

Using photoaffinity labeling, we have identified a region inmammalian farnesyl-proteintransferase (FPTase) involved in substrate recognition. Thephotolabel used (Compound 1) is a peptidecontaining the photoactive amino acid p-benzoylphenylalanine(Bpa). Upon exposure to UV light,Compound 1 inhibits FPTase activity in a time- andconcentration-dependent manner. Photoinhibition ofFPTase activity by Compound 1 is prevented by adding H-Ras to thereaction mixture, indicating thatlabeling is targeted to the enzyme active site. We used peptidemapping by HPLC, Edman sequencing,and matrix-assisted time-of-flight (MALDI-TOF) mass spectrometry toidentify the site of interactionwith radiolabeled Compound 1. These experiments indicate that aspecific region of the ages/gifchars/alpha.gif" BORDER=0> subunit of theenzyme, Asp110-Arg112, is involved in substrate binding and suggestthat Glu111 is likely to be theresidue covalently modified by the photoaffinity label. Sequencealignments between yeast and mammalianFPTases reveal that Glu111 is conserved. The implications of thisfinding are discussed in light of previousmutagenesis studies on FPTase.