STUDIES ON ENZYMATIC-ACTIVITY AND CONFORMATIONAL STABILITY OF MUSCLE ACYLPHOSPHATASE MUTATED AT CONSERVED LYSINE RESIDUES

An oligonucleotide-directed mutagenesis study was carried out on the f ive acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to elim inate the ambiguity arising from the presence of a sulfate ion, an enz yme competitive inhibitor, bound to lysine 32 and 68 in the crystal st ructure of the erythrocyte isoenzyme, Furthermore, previous kinetic st udies suggested the presence of residues with pK(a) = 7.9 and 11, tent atively identified as two lysines, The kinetic parameters for the muta nts under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues a re involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism ind icate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy av erages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contri bution to the stability of the native structure.