STRUCTURAL AND KINETIC INVESTIGATIONS ON THE 15-21 AND 42-45 LOOPS OFMUSCLE ACYLPHOSPHATASE - EVIDENCE FOR THEIR INVOLVEMENT IN ENZYME CATALYSIS AND CONFORMATIONAL STABILIZATION

The structural and catalytic importance of the 15-21 and 42-45 loop re sidues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving cons erved residues of the two loops have been prepared and characterized f or structural, kinetic, and stability features by using different spec troscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homolog ous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 a nd the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of t he 15-21 loop mutants. In particular, the Gly15Ala mutant is completel y inactive and displays a native-like overall fold, indicating that th e correct geometry of the 15-21 loop is an essential requisite for opt imal enzymatic catalysis. Instead, the Gly45Ala mutant, though reveali ng unchanged catalytic properties, shows a considerably reduced confor mational stability, as judged by circular dichroism and H-1 NMR spectr oscopy. This finding confirms previous results relative to Thr42 and T hr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underl ining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.