Abstract
Photoreactive analogues of prenyl diphosphates have been useful in studying prenyltransferases. The effectiveness of analogues with different chain lengths as probes of recombinant human protein prenyltransferases is established here. A putative geranylgeranyl diphosphate analogue, 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate (DATFP-FPP), was the best inhibitor of both protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PFFT-I). Shorter photoreactive isprenyl diphosphate analogues with geranyl and dimethylallyl moieties and the DATFP-derivative of farnesyl monophosphate were much poorer inhibitors. DATFP-FPP was a competitive inhibitor of both PFT and PGGT-I with Ki values of 100 and 18 nM, respectively. [32P]DATFP-FPP specifically photoradiolabelled the beta-subunits of both PFT and PGGT-I. Photoradiolabelling of PGGT-I was inhibited more effectively by geranylgeranyl diphosphate than farnesyl diphosphate, whereas photoradiolabelling of PFT was inhibited better by farnesyl diphosphate than geranylgeranyl diphosphate. These results lead to the conclusions that DATFP-FPP is an effective probe of the prenyl diphosphate binding domains of PFT and PGGT-I. Furthermore, the beta-subunits of protein prenyltransferases must contribute significantly to the recognition and binding of the isoprenoid substrate.
Highlights
Prenylated proteins have an important role in cellular regulation, a description of their structure/function relationships and post-translational processing has been of great interest [1,2,3]
The monophosphate of DATFP-farnesol (DATFP-FMP) was a much poorer inhibitor than the corresponding diphosphate with inhibitory properties similar to DATFP-GPP. The effectiveness of these inhibitors on hPGGT-I was similar to that exhibited with hPFT, except that DATFP-GPP was no better an inhibitor than the shorter analogue DATFP-DMAPP (Fig. lB)
HPGGT-I was competitively inhibited by DATFP-farnesyl diphosphate (FPP) with a K, value of 18 nM, whereas the estimated Km value of 16 nM was observed for its isoprenoid substrate, geranylgeranyl diphosphate (GGPP) (Fig. 2B)
Summary
19035-19040, 1995 Printed in U.S.A. Photoreactive Analogues of Prenyl Diphosphates as Inhibitors and Probes of Human Protein Farnesyltransferase and Geranylgeranyltransferase Type 1*. The substrate specificity of prenyltransferases which recognize the -C-AI-A2-X motif has been well established and many inhibitors of these enzymes have been described [16,17,18,19,20], the full extent of involvement of the protein subunits or their respective amino acid residues in the prenylation reaction has not yet been elucidated for any of these prenyltransferases. The radioactive photoreactive prenyl diphosphate probes labeled only the f3-subunits of these prenyltransferases and the labeling could be inhibited by the natural substrate for each enzyme, no evidence was presented to unequivocally demonstrate that the photoprobe binds to the active site.
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