EVIDENCE BY SITE-DIRECTED MUTAGENESIS THAT ARGININE-203 OF THERMOLYSIN AND ARGININE-717 OF NEPRILYSIN (NEUTRAL ENDOPEPTIDASE) PLAY EQUIVALENT CRITICAL ROLES IN SUBSTRATE HYDROLYSIS AND INHIBITOR BINDING

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, s ite-directed mutagenesis studies have shown that its active site resem bles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active sire residue of thermolysin, Arg-203, is invol ved in inhibitor binding by forming hydrogen bonds with the carbonyl g roup of a residue in the P-1' position and also participates in a hydr ogen bond network involving Asp-170. Sequence alignment data shows tha t Arg-717 of neprilysin could play a similar role to Arg-203 of thermo lysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in k(cat)/K-m values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in k(cat). The K-j values of several inhibitors were a lso increased for the mutated enzymes. In addition, the replacement. o f Asp-170 of thermolysin by Ala residue resulted in a decrease in k(ca t)/K-m of 220-fold. The results, coupled with a molecular modeling stu dy, suggest that Arg-717 of neprilysin corresponds to Arg-203 of therm olysin and that in both enzymes a hydrogen bond network exists, involv ing His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.