Purification and characterization of glycerate kinase from the thermoacidophilic archaeon Thermoplasma acidophilum:: An enzyme belonging to the second glycerate kinase family

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at 59 degrees C and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus it is known to metabolize glucose by the non-phosphorylated Entner-Doucloroff (nED) pathway. in the course of these studies, the specific activities of glyceralclehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49 kDa and followed Michaelis-Menten kinetics with K-m values of 0.56 and 0.32 mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at 70 degrees C. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as Mn2+, Co2+, Ni2+, Zn2+, Ca2+, and Sr2+, were substituted for Mg2+, the enzyme activities were less, than 10% of that obtained in the presence of Mg2+. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.