7] Phosphoglycerate kinase-triose-phosphate isomerase complex from Thermotoga neapolitana

Abstract

Publisher Summary Phosphoglycerate kinase (PGK) is one of two enzymes that conserves energy by substrate level phosphorylation in glycolysis. The reversible reaction catalyzed by phosphoglycerate kinase involves the transfer of the acyl phosphate of 1,3-diphosphoglycerate (1,3-DPG) to ADP forming ATP and 3-phosphoglycerate (3-PGA). Because this enzyme is highly conserved in both sequence and function among Bacteria, Archaea, and Eukarya, phosphoglycerate kinase is an excellent candidate for studying the determinants of thermal stability of enzymes. Therefore, the structure of PGK among members of the Thermotogales , a bacterial family of hyperthermophiles, is of considerable interest. The fus gene encoding a protein with both 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI) activities was cloned from T. maritima . Later, the tpi gene alone was cloned and expressed in Escherichia coli as a monomer. A crystallographic analysis of the cloned fusion protein allowed comparisons with mesophilic homologs in attempts to reveal sequence elements that confer thermal stability. This chapter describes the methods used to study this fusion protein and those methods that are of general applicability to identify other such genes.