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Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis
K E Hightower1, S De, C Weinbaum
1Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, NC 27710, U.S.A.
Abstract:
Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.
Insights
The positive charge of Lysine 164 in protein farnesyltransferase (FTase) is crucial for binding substrates and forming the enzyme-farnesyl diphosphate-peptide ternary complex. This residue
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Protein farnesyltransferase (FTase) is a therapeutic target due to its role in oncogenic Ras protein prenylation and tumor formation.
- Structural studies identified specific FTase residues potentially involved in substrate binding and catalysis.
- Understanding FTase mechanism is key for developing novel anti-cancer drugs.
Purpose of the Study:
- To investigate the role of Lysine 164 (Lys164) in the alpha subunit of FTase.
- To determine the significance of the positive charge at Lys164 for substrate binding and catalytic activity.
- To elucidate the contribution of Lys164 to the formation of the enzyme-farnesyl diphosphate-peptide ternary complex.
Main Methods:
- Site-directed mutagenesis was used to create mutant FTase enzymes with arginine or alanine substitutions at Lys164.
- Kinetic assays were performed to compare the substrate binding and catalytic rates of wild-type and mutant FTase enzymes.
- Analysis focused on association rate constants, binding affinities, and observed rate constants for product formation.
Main Results:
- Mutation of Lys164 significantly affected the association rate constant and binding affinity for a CaaX peptide substrate.
- The positive charge of Lys164 was found to be essential for the formation of the E.FPP.peptide ternary complex.
- Mutations at Lys164 substantially decreased the rate constant for product formation without altering the catalytic mechanism.
Conclusions:
- The positive charge and precise positioning of Lys164 in FTase are critical for efficient substrate binding and catalysis.
- Lys164 plays a vital role in the formation of the ternary complex necessary for FTase activity.
- These findings provide valuable insights into FTase mechanism, aiding in the rational design of FTase inhibitors for cancer therapy.