The H159A mutant of yeast enolase 1 has significant activity

The function of His159 in the enolase mechanism is disputed, Recently, Vina rov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A muta nt of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appea red to be correctly folded, according to its CD spectrum, tryptophan fluore scence, and binding of metal ion and substrate. We prepared H159A enolase u sing a multicopy plasmid and expressed the enzyme in yeast, Our preparation s of H159A enolase have 0.2-0.4% of the native activity under standard assa y conditions and are further activated by Mg2+ concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 land enolase 2) are inhibi ted by such Mg2+ concentrations. It is possible that His159 is necessary fo r correct folding of the enzyme and that expression in E. coli leads to lar gely misfolded protein, (C) 2000 Academic Press.