Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state.
Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.
Dion
N
N
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
Le Moual
H
H
Crine
P
P
Boileau
G
G
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014162:Transfection
D006639:Histidine; D016308:Enkephalin, Leucine-2-Alanine; C030279:enkephalinamide-Leu, Ala(2)-; D004247:DNA; D015260:Neprilysin; D004047:Diethyl Pyrocarbonate