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  4. Reaction mechanism of trans-type octaprenyl pyrophosphate synthase from E. coli and Thermotoga maritima
 
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Reaction mechanism of trans-type octaprenyl pyrophosphate synthase from E. coli and Thermotoga maritima

Date Issued
2005
Date
2005
Author(s)
Chang, Keng-Ming
DOI
en-US
URI
http://ntur.lib.ntu.edu.tw//handle/246246/52798
Abstract
ABSTRACT Octaprenyl pyrophosphate synthase (OPPS) catalyzes consecutive condensation reactions of one molecule of farnesyl pyrophosphate (FPP) with five molecules of isopentenyl pyrophosphates (IPP) to generate C40 octaprenyl pyrophosphate (OPP). The octaprenyl group constitutes the side chain of ubiquinone or menaquinone, an essential component for ATP synthesis. Two DDXXD motifs conserved among all the trans-prenyltransferases were suspected to bind FPP and IPP substrates via Mg2+. However, two sulfate ions S1 and S2 were found binding in the first DDXXD and a positively-charged pocket in the active site of T. maritima OPPS, respectively. To examine the substrate-binding mode, we built a structural model for E. coli OPPS based on its sequence homology to the T. maritima OPPS and performed site-directed mutagenesis. The amino acids D84, D85, and D88 of the first DDXXD and R93, which presumably bind the first sulfate ion, are essential for FPP binding and catalysis. The K45, R48, H77, and R94, which presumably bind the other sulfate ion, are more important for IPP binding and catalysis. These results suggest a substrate-binding mode, in which FPP binds to the first DDXXD motif and IPP binds to the S2 site. This conclusion is also supported by the crystal structures of the mutant T. maritima OPPS. By using fluorescent stopped-flow method, the kinetic scheme for the OPPS reaction was determined. OPPS binds FPP and IPP in a random order and the product release represents the rate limiting step. The trapping of the reaction intermediate, pH profile support the ionization-condensation-elimination mechanism for OPPS reaction. We also determined the pKa of essential amino acids derived from pH profile studies of wild type and H77D and H77Q mutants and found that H77 might act as a general base in the OPPS reaction. Moreover, the role of metal ion in catalysis and the substrate specificity, as well as the computer modeling of the inhibitors were also studied.
Subjects
反式─八異戊二烯焦磷酸合成酶
trans-type octaprenyl pyrophosphate synthase
Type
other
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