Yeast protein farnesyltransferase. Site-directed mutagenesis of conserved residues in the beta-subunit.
Protein prenyltransferases catalyze the posttranslational modification of cysteines by isoprenoid hydrocarbon chains. A protein farnesyltransferase (PFTase) and a protein geranylgeranyltransferase (PGGTase-I) alkylate cysteines in a CaaX C-terminal tetrapeptide sequence, where a is usually an aliphatic amino acid and X is an amino acid that specifies whether a C15 farnesyl or C20 geranylgeranyl moiety is added. A third enzyme, PGGTase-II, adds geranylgeranyl groups to both cysteines at the C-terminus of Rab proteins. All three enzymes are Zn2+ metalloproteins and also require Mg2+ for activity. The protein prenyltransferases are heterodimers. PFTase and PGGTase I contain identical alpha-subunits and distinctive beta-subunits, which are responsible for the differences in substrate selectivity seen for the two enzymes. The subunits in PGGTase-II are similar, but not identical, to their counterparts in the other two enzymes. An alignment of amino acid sequences for the beta-subunits of all three enzymes shows five regions of high similarity. Thirteen of the conserved polar and charged residues in yeast PFTase were selected for substitution by site-directed mutagenesis. Kinetic studies revealed a subset of five enzymes, R211Q, D307A, C309A, Y310F, and H363A, with substantially reduced catalytic constants (kcat). Metal analyses of wild-type enzyme and the five least reactive mutants showed that the substitutions had compromised Zn2+ binding in the D307A, C309A, and H363A enzymes.
Dolence
J M
JM
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Rozema
D B
DB
Poulter
C D
CD
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D016415:Sequence Alignment; D014166:Transferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D015032:Zinc