M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue

Abstract

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in 16 Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide 17 destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-18 shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined 19 by seven conserved residues. Five of them are involved in metal ion binding which is important 20 to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, 21 is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The 22 seventh residue is an aspartate whose function remains poorly understood. This aspartate 23 residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. 24 It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

“…We study here the 468 kDa large tetrahedral aminopeptidase TET2 from the hyperthermophilic archaeon Pyrococcus horikoshii , a member of the metallo-peptidase family M42. Archaeal TET aminopeptidases and homologous structures in other organisms 12 , 13 assemble to dodecameric tetrahedral structures, encapsulating twelve Zn 2 active sites within a large hollow lumen with a diameter of ca. 60 Å 14 – 17 (Fig.…”

“…The proposed catalytic mechanism is common to binuclear metallo-aminopeptidases, such as Leucyl-aminopeptidase (LAP) and AAP 12 , 22 , 23 . In this mechanism, peptide bond cleavage proceeds via the activation (deprotonation) of a water molecule that bridges the zinc ions.…”

Substrate Characterization for Hydrolysis of Multiple Types of Aromatic Esters by Promiscuous Aminopeptidases

“…Research indicates that the promiscuity of enzyme catalysts is implicit in Ohno’s evolutionary model, and comparative studies of functional divergence within promiscuous enzyme families have been used successfully to provide insight into the evolution of function in various protein families. , Thus, enzyme promiscuity can be regarded as the starting point for the emergence of new functional activities in enzymes in nature. , Both aminopeptidases and carboxylesterases belong to the α/β hydrolase superfamily. Despite differences in catalytic mechanisms, these enzymes share similar backbone structures, catalytic active sites, and catalytic triads (Glu-Ser-His/Asp). , Moreover, both enzymes involve similar transition states in their catalyzed reactions. , Additionally, the protease-associated (PA) domain of aminopeptidase serves as a scaffold for binding large substrates during catalysis, enlarging the size of the binding pocket . Similarly, carboxylesterases that catalyze the hydrolysis of aromatic esters have a wider and shallower catalytic cavity, lacking a “lid” at the fissure of the active site, facilitating the entry of large molecular substrates.…”

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

“…We study here the 468 kDa large tetrahedral aminopeptidase TET2 from the hyperthermophilic archaeon Pyrococcus horikoshii, a member of the metallo-peptidase family M42. Archaeal TET aminopeptidases and homologous structures in other organisms (12,13) assemble to dodecameric tetrahedral structures, encapsulating twelve Zn 2 active sites within a large hollow lumen with a diameter of ca. 60 Å (14-17) (Fig.…”

“…(15) The proposed catalytic mechanism is common to binuclear metallo-aminopeptidases, such as Leucyl-aminopeptidase (LAP) and AAP. (12,22,23) In this mechanism, peptide bond cleavage proceeds via the activation (deprotonation) of a water molecule that bridges the zinc ions. The water molecule donates a proton to a conserved glutamate (E212 in TET2), and the remaining hydroxyl group then attacks the peptide bond of the substrate (15,22,24).…”