Reaction kinetic pathway of the recombinant octaprenyl pyrophosphate synthase from Thermotoga maritima: how is it different from that of the mesophilic enzyme.
Octaprenyl pyrophosphate synthase (OPPs) catalyzes the chain elongation of farnesyl pyrophosphate (FPP) via consecutive condensation reactions with five molecules of isopentenyl pyrophosphate (IPP) to generate all-trans C40-octaprenyl pyrophosphate. The polymer forms the side chain of ubiquinone that is involved in electron transport system to produce ATP. Our previous study has demonstrated that Escherichia coli OPPs catalyzes IPP condensation with a rate of 2 s(-1) but product release limits the steady-state rate at 0.02 s(-1) [Biochim. Biophys. Acta 1594 (2002) 64]. In the present studies, a putative gene encoding for OPPs from Thermotoga maritima, an anaerobic and thermophilic bacterium, was expressed, purified, and its kinetic pathway was determined. The enzyme activity at 25 degrees C was 0.005 s(-1) under steady-state condition and was exponentially increased with elevated temperature. In contrast to E. coli OPPs, IPP condensation rather than product release was rate limiting in enzyme reaction. The product of chain elongation catalyzed by T. maritima OPPs was C40 and the rate of its conversion to C45 was negligible. Under single-turnover condition with 10 microM OPPs-FPP complex and 1 microM IPP, only the C20 was formed rather than C20-C40 observed for E. coli enzyme. Together, our data suggest that the thermophilic OPPs from T. maritima has lower enzyme activity at 25 degrees C, higher product specificity, higher thermal stability and lower structural flexibility than its mesophilic counterpart from E. coli.
Kuo
Tun-Hsun
TH
Institute of Biochemical Sciences, National Taiwan University, Taipei 10098, Taiwan.
Liang
Po-Huang
PH
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology*
D019883:Alkyl and Aryl Transferases; C048987:octaprenyl pyrophosphate synthetase