Phosphoisoprenoid binding specificity of geranylgeranyltransferase type II.
Geranylgeranyltransferase type II (GGTase-II) modifies small monomeric GTPases of the Rab family by attaching geranylgeranyl moieties onto two cysteines of their C-terminus. We investigated to what extent GGTase-II discriminates between its native substrate geranylgeranyl pyrophosphate (GGpp) and other phosphoisoprenoids, including farnesyl pyrophosphate (Fpp). On the basis of a novel fluorescent assay, we demonstrated that GGpp binds to GGTase-II with an affinity of 8 +/- 4 nM, while Fpp is bound less strongly (K(d) = 60 +/- 8 nM). Analysis of the binding kinetics of four different phosphoisoprenoids indicated that in all cases association is rapid, with rate constants in the range of 0.15 nM(-1) s(-1). In contrast, the dissociation rates differed greatly, depending on the phosphoisoprenoid used, with weak binding substrates generally displaying an increased rate of dissociation. The affinity of GGpp and Fpp for GGTase-II was also determined in the presence of the Rab7-REP-1 complex. The affinity for GGpp was essentially unaffected by the presence of the complex; Fpp on the other hand bound less strongly to the GGTase-II under these conditions, resulting in a K(d) of 260 +/- 60 nM. In vitro prenylation experiments were used to establish that Fpp not only does bind to GGTase-II but also is transferred with an observed rate constant of 0.082 s(-1) which is very similar to that of GGpp. The implications of the low level of discrimination by GGTase-II for the in vivo specificity of the enzyme and the use of farnesyltransferase inhibitors in anti-cancer therapy are discussed.
Thomä
N H
NH
Max-Plank-Institute for molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Iakovenko
A
A
Owen
D
D
Scheidig
A S
AS
Waldmann
H
H
Goody
R S
RS
Alexandrov
K
K
D048868:Adaptor Proteins, Signal Transducing; D000345:Affinity Labels / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017368:Protein Prenylation; D012717:Sesquiterpenes; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism; D020691:rab GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D000345:Affinity Labels; C081787:CHM protein, human; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D062367:ortho-Aminobenzoates; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C075607:Rab geranylgeranyltransferase; C083291:geranylgeranyltransferase type-I; D020691:rab GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate