H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate.
The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras binds to FTase in the microM range (KD = 4 microM) at physiological pH; however, the peptide affinity is enhanced approximately 70-fold in a ternary complex with an enzyme-bound farnesyl diphosphate (FPP) analogue, indicating that the two substrates bind synergistically. The pH dependence of substrate binding was also investigated, and two ionizations were observed: for the ternary complex, the pKa values are 8.1, reflecting ionization of the thiol of the free peptide, and 6.4. The pH dependence of the ligand-metal charge-transfer band in the optical absorption spectra of a Co2+-substituted FTase ternary complex suggests that a metal-coordinated thiol ionizes with a pKa of 6.3. These data indicate that metal coordination of the peptide sulfur with the zinc ion in FTase lowers the pKa of the thiol resulting in formation of a bound thiolate at physiological pH.
Hightower
K E
KE
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Huang
C C
CC
Casey
P J
PJ
Fierke
C A
CA
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D003035:Cobalt / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
D003619:Dansyl Compounds; D009842:Oligopeptides; D013438:Sulfhydryl Compounds; D003035:Cobalt; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D016283:Proto-Oncogene Proteins p21(ras); D015032:Zinc; D003545:Cysteine