Among the trans-PTs, short-chain prenyltransferases (SC-PTs)
catalyze the sequential head-to-tail addition of up to three molecules of IPP to one molecule of DMAPP producing C10 geranyl
diphosphate (GPP), C15 farnesyl diphosphate (FPP) or C20 geranylgeranyl diphosphate (GGPP) [2, 3] (Fig. 1). This enzymatic
reaction requires two highly conserved domains, referred to as first
aspartate-rich motif (FARM, DDx 2-4 D) and second aspartate-rich
motif (SARM, DDx 2 D), that are involved in substrate binding [9–
14] and catalysis using Mg
2+ as cofactor [15]. The sequential
addition of IPP molecules to the isoprenyl diphosphate product
releases a pyrophosphate (PPi) molecule per IPP unit added. This
process takes place in the elongation pocket of the SC-PT, a hydrophobic cavity that controls the hydrocarbon tail length by the size
of the side chain of some amino acid residues located on the
N-terminal side of the FARM. In most cases, the fifth amino acid
upstream to this motif is the responsible of the isoprenyl product
chain length [16–22]. However, other residues could also play a
role in controlling the number of IPP condensations during product elongation [3].
Despite all the studies highlighting the role of the elongation
pocket on final product length, predictions are difficult and highly
sensitive analytical methods are still required to determine the
actual product of uncharacterized SC-PTs. Radioactive enzymatic
assays followed by the hydrolysis of the products require specialized
equipment for the measurements such as radio-gas chromatography (radio-GC), radio-high-performance liquid chromatography
(radio-HPLC), thin layer chromatography (TLC), or liquid scintillation counting (LSC) [22–28]. These methods, however, often fail
to conclusively demonstrate the chain length (i.e., identity) of the
product. Moreover, they are extremely time-consuming when used
to calculate the enzymatic kinetics of the tested SC-PT. Nonradioactive methods based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) have been developed to
accurately determine isoprenyl diphosphate metabolites in a much
more precise and faster way [29–31]. However, they are still complex and expensive for the enzymatic characterization of SC-PTs
(i.e., for the calculation of their optimal pH or kinetic parameters
such as V max and K m ).
Here we describe an easy and inexpensive spectrophotometric
protocol to enzymatically characterize previously identified SC-PTs
using purified enzyme preparations. The described method is an
adaptation of the commercial EnzChek Pyrophosphate Assay Kit
(E-6645), that allows the detection of the PPi released by a biochemical reaction. The PPi produced in the reaction mix is converted into two molecules of inorganic phosphate (Pi) by an
inorganic pyrophosphatase. The Pi then reacts with the substrate
2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) by a
purine nucleoside phosphorylase (PNP) producing ribose
Determination of GGPP Synthase Activity
29
catalyze the sequential head-to-tail addition of up to three molecules of IPP to one molecule of DMAPP producing C10 geranyl
diphosphate (GPP), C15 farnesyl diphosphate (FPP) or C20 geranylgeranyl diphosphate (GGPP) [2, 3] (Fig. 1). This enzymatic
reaction requires two highly conserved domains, referred to as first
aspartate-rich motif (FARM, DDx 2-4 D) and second aspartate-rich
motif (SARM, DDx 2 D), that are involved in substrate binding [9–
14] and catalysis using Mg
2+ as cofactor [15]. The sequential
addition of IPP molecules to the isoprenyl diphosphate product
releases a pyrophosphate (PPi) molecule per IPP unit added. This
process takes place in the elongation pocket of the SC-PT, a hydrophobic cavity that controls the hydrocarbon tail length by the size
of the side chain of some amino acid residues located on the
N-terminal side of the FARM. In most cases, the fifth amino acid
upstream to this motif is the responsible of the isoprenyl product
chain length [16–22]. However, other residues could also play a
role in controlling the number of IPP condensations during product elongation [3].
Despite all the studies highlighting the role of the elongation
pocket on final product length, predictions are difficult and highly
sensitive analytical methods are still required to determine the
actual product of uncharacterized SC-PTs. Radioactive enzymatic
assays followed by the hydrolysis of the products require specialized
equipment for the measurements such as radio-gas chromatography (radio-GC), radio-high-performance liquid chromatography
(radio-HPLC), thin layer chromatography (TLC), or liquid scintillation counting (LSC) [22–28]. These methods, however, often fail
to conclusively demonstrate the chain length (i.e., identity) of the
product. Moreover, they are extremely time-consuming when used
to calculate the enzymatic kinetics of the tested SC-PT. Nonradioactive methods based on liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) have been developed to
accurately determine isoprenyl diphosphate metabolites in a much
more precise and faster way [29–31]. However, they are still complex and expensive for the enzymatic characterization of SC-PTs
(i.e., for the calculation of their optimal pH or kinetic parameters
such as V max and K m ).
Here we describe an easy and inexpensive spectrophotometric
protocol to enzymatically characterize previously identified SC-PTs
using purified enzyme preparations. The described method is an
adaptation of the commercial EnzChek Pyrophosphate Assay Kit
(E-6645), that allows the detection of the PPi released by a biochemical reaction. The PPi produced in the reaction mix is converted into two molecules of inorganic phosphate (Pi) by an
inorganic pyrophosphatase. The Pi then reacts with the substrate
2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) by a
purine nucleoside phosphorylase (PNP) producing ribose
Determination of GGPP Synthase Activity
29