Leucine aminopeptidase, active site structure

N Strater, WN Lipscomb (1995) Two-metal ion mechanism of bovine lens leucine aminopeptidase active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography, Biochemistry 34 14792-14800... 

Fig. 22. The active site structure of phospholipase C and a proposed structure for the leucine aminopeptidase site.

Figure 6 The active site structures of (left) bovine lens leucine aminopeptidase (pdb ILAM) and (right) Aeromonas proteolytica leucine aminopeptidase (pdb 1 AMP). The zinc ions are in dark gray, carbon atoms in light gray, oxygen atoms in white, and nitrogen atoms in black. Hydrogen bonding interactions are indicated...

While Co " competes with Zn for binding at both sites in bovine lens leucine aminopeptidase, Mg " " competes for only one of the sites. When cobalt(ii) is substituted at site 1, the specific activity with L-leucine-p-nitroanilide is increased by more than ten-fold. Cobalt(ii) has been replaced for zinc in thermolipin and simultaneously terbium(iii) for calcium the observed visible fluorescence (excitation at 280 nm) provided quantum yield measurements which indicated that the terbium-cobalt distance was 13.7 A, in good agreement with the Ca—Zn distance determined from a recent crystal structure. Vallee and co-workers suggest that this technique... 

Bovine leucine aminopeptidase (BLAP) is another classical binuclear metalloprotease that belongs to the zinc a,/3-hydrolase superfamily of enzymes and is topologically similar to AAP." However, the overall location of the binuclear metal center and the coordination of the individual zinc ions differ from those observed for AAP. Crystal structures of BLAP indicate that the two zinc ions in the active-site binuclear metal center are closer together (2.3—3.0 A separation) than in the AAP. The two zinc sites of the BLAP binuclear metal center are each a five-coordinated unsymmetrical species with what is best described as an octahedral... 

A major part of the work on the structural requirements for binding of inhibitors to the active site of either aminopeptidase M or aminopeptidase Mil has been conducted using bestatin (see Figure 6.3A) as the model inhibitor and interpreted from the known structure-activity relationships of bestatin analogues towards leucine-aminopeptidase [43]. 

Because all of these important residues for the blLAP catalytic mechanism are found to be fully conserved in the E. coli [34] and Rickettsia prowazekii [37] pepA-encoded leucine aminopeptidases, a sequence alignment has been performed to see whether these residues are also present in the P. putida L-aminopeptidase. This indeed appears to be the case (see Fig. 4). Interestingly, it was observed that these residues were fully conserved in all currently known leucine aminopeptidases that are significantly homologous to the P. putida L-aminopeptidase. Because of these apparent similarities in (1) overall primary structure (Table 4) and (2) key active site residues among the members of the leucine... 

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