Yeast protein farnesyltransferase. Site-directed mutagenesis of conserved residues in the beta-subunit

被引:31
作者
Dolence, JM [1 ]
Rozema, DB [1 ]
Poulter, CD [1 ]
机构
[1] UNIV UTAH,DEPT CHEM,SALT LAKE CITY,UT 84112
关键词
D O I
10.1021/bi970039p
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 C-15 farnesyl or C-20 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 (k(cat)) 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.
引用
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页码:9246 / 9252
页数:7
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