Purification and Properties of Phosphoglycerate Kinase from Thermus thermophilus Strain HB8

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Abstract
A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10–20 min at 79°C. Catalytic properties such as pH optimum (pH 6–8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0. The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% α-helix and 11 % pleated sheet from the circular dichroic spectrum of the enzyme protein. The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.