PMC

Rate dependence of PGI purified from P. furiosus on the glucose-6-phosphate concentration at 80°C. The insert shows a double reciprocal plot of the rates versus the corresponding substrate concentrations. Enzyme activity was measured in the discontinuous assay system (see Materials and Methods). The assay mixture contained 0.18 μg of enzyme.

Multiple sequence alignment of deduced amino acid sequences of PGI from P. furiosus and of putative PGI from P. horikoshii () (PH1956) and P. abysii (Genoscope database; see above) (PAB1199). The sequence of PH1956 (192 amino acids) was truncated by 3 amino acids due to the identification of a putative ribosome binding site (TGGTGA) between bp −3 to −8 upstream of the start codon ATG.

Effect of temperature on the specific activity of the PGI purified from P. furiosus. (A) Temperature dependence of the specific activity. (B) Arrhenius plot of the same data. Enzyme activity was measured in the direction of glucose-6-phosphate formation using either the continuous assay for temperatures below 50°C (▵) or the discontinuous assay system for temperatures above 50°C (●) (see Materials and Methods). The assay mixture contained 0.35 μg of enzyme.

Multiple sequence alignment of the PGI superfamily of bacteria and eucarya. Deduced amino acid sequences of B. stearothermophilus B (), E. coli (), pig (), rabbit (), and human () are aligned. In addition, the amino acid sequence of the hypothetical PGI of the archaeon M. jannaschii (MJ1605) is included. The two PGI signature patterns [DENS]-X-[LIVM]-G-G-R-[FY]-S-[LIVMT]-X-[STA]-[PSAC]-[LIVMA]-G- and [GS]-X-[LIVM]-[LIVMFYW]-XXXX-[FY]-[DN]-Q-X-G-V-E-X-X-K are highlighted by boxes. Amino acids that constitute putative substrate-binding sites in accordance with the crystal structure of B. stearothermophilus PGI () are shaded.

Thermostability of PGI purified from P. furiosus enzyme (1.8 μg) was incubated in 50 μl of 50 mM potassium phosphate buffer, pH 7.0, at 70°C (□), 80°C (▵), 90°C (●), and 100°C (○). At the times indicated, 15-μl aliquots were withdrawn and assayed for remaining activity at 55°C in the direction of glucose-6-phosphate formation. One hundred percent activity corresponded to the specific activity of PGI of 30 U/mg.

Purification of PGI from P. furiosus (A) and of recombinant PGI from transformed E. coli (B) as analyzed by SDS-PAGE. Protein was denatured in SDS and separated in 14% (A) or 12% (B) slab gels (8 by 7 cm) (), which were stained with Coomassie brilliant blue R 250. (A) Lanes 1 and 7, molecular mass standards (Sigma), in kilodaltons; lanes 2 to 6, analysis of PGI after various steps of the purification procedure (lane 2, 100,000 × g supernatant; lane 3, Q Sepharose; lane 4, phenyl-Sepharose; lane 5, Superdex 200; lane 6, Uno Q1). (B) Lane 1, molecular mass standards (Sigma); lane 2, purified recombinant PGI.