The H159A mutant of yeast enolase 1 has significant activity.
The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138-12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2-0.4% of the native activity under standard assay conditions and are further activated by Mg(2+) concentrations above 1 mM to 1-1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg(2+) concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein.
Brewer
J M
JM
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, 30602, USA. brewer@bmbiris.bmb.uga.edu
Holland
M J
MJ
Lebioda
L
L
D019943:Amino Acid Substitution / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation / Q000235:genetics*; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D006639:Histidine; D010751:Phosphopyruvate Hydratase; D008274:Magnesium