Bovine lens leucine aminopeptidase

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... 

H Kim, WN Lipscomb (1994) Structure and mechanism of bovine lens leucine aminopeptidase, Adv Enzymol Relat Areas Mol Biol 68 153-213... 

The structure of bovine lens leucine aminopeptidase 104) has also been determined by X-ray crystallography but in this case the resolu-... 

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... 

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...

Figure 7 Drawing of the complex of bovine lens leucine aminopeptidase with the transition state analogue L-leucinal. Note the coordination mode of the gem-diolate in the bottom center. The spheres labeled 1 and 2...

Bovine lens leucine aminopeptidase is a hexameric enzyme. Each of its six identical subunits exhibits independent catalytical activity and contains two zinc ions in the active site. These metal ions are essential for catalytic activity because both participate in substrate binding and activation, including a possible role in the activation of the nucleophile [43]. 

Tabie 5 Role of Different Amino Acid Residues of the Bovine Lens Leucine Aminopeptidase in the Two-Metal Ion Catalytic Mechanism [29,33,36]... 

Figure 4 Sequence alignment of the deduced amino acid sequence of P. putida PepA with the primary sequences of E. coli aminopeptidase A, Rickettsia prowazekii aminopeptidase A, and bovine eye lens leucine aminopeptidase (blLAP). The bottom line is the consensus sequence, which was generated by the multiple sequence alignment algorithm Clustal W. Residues in boldface and marked with an arrow represent residues of the blLAP that have been shown to be involved in metal coordination and/or inhibitor binding.

A Taylor, M Daims, J Lee, T Surgenor (1982) Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins, Curr Eye Res 2 47-56... 

The direct acylation and methylation of the free 01-NH2 groups of proteins have been proposed to be useful in providing resistance toward proteolytic attacks. Although the basis for this explanation is not always readily apparent from the known specificities of proteases, it may be valid in some cases. Thus, the acetylated N-terminus of a-crystallin, the major protein found in eye lens (17), is presumably important for the protein to survive in an environment rich in leucine aminopeptidase. On the other hand, it is difficult to rationalize that the acetylated N-terminus of bovine pancreatic a-amylase is in any way responsible for the fact that the enzyme is exceedingly stable against tryptic and chymotryptic digestion (18). The function of the acetylation is, in this case, as obscure as is the basis on which a-amylase is selected for acetylation among the many non-acetylated companion pancreatic proteins. 

This discussion of the metalloexopeptidases has focused on the general role of these enzymes in the conversion of dietary proteins into amino acids. In particular, the apparent synergistic relationship which the pancreatic carboxypeptidases have with the major endopeptidases, trypsin, chymotrypsin, and pepsin, in order to facilitate formation of essential amino acids has been stressed. The chemical characteristics, metalloenzyme nature, and mechanistic details of a representative of each class of exopeptidase have been presented. Leucine aminopeptidase from bovine lens was shown to be subject to an unusual type of metal ion activation which may be representative of a more general situation. Carboxypeptidase A of bovine pancreas was discussed in terms of its three-dimensional structure, the implications of x-ray crystallography to mecha ... 

Aminopeptidases are counterparts to carboxypeptidases, removing N-terminal amino acids. However, unlike the carboxypeptidases, they contain dinuclear zinc sites. They fall into two groups, the first of which includes the leucine aminopeptidase from bovine lens, while the second includes the leucine aminopeptidases AAP from Aeromonas proteolytica and SAP from Streptomyces griseus (Figure 12.15). The mechanism of the AAP enzyme has been well studied, and may well represent a general catalytic mechanism for peptide hydrolysis by metal-lopeptidases with a cocatalytic active site.ki. 

This section will focus on the structure and properties of the two best characterized examples of aminopeptidases that contain dinuclear zinc centers, both of which happen to be leucine aminopeptidases the enzymes from bovine lens (blLAP) and the marine bacterium Aeromonas proteo-lytica (AAP). Despite the fact that they catalyze the same reaction with comparable efficiencies,... 

Leucine aminopeptidase EC 3.4.11.1 Leu-X- (AA-X-) Bovine and pig lens and kidney Activated by Mg, Mn basic pH optimum chromogenic substrates not hydrolysed inhib. by 1,10 phenanthroline, acti-nonin, bestatin 6 subunits of M, 53000. 

Leucine aminopeptidase preferentially hydrolyses peptide bonds adjacent to an Al-terminal residue that carries a large hydrophilic side chain, in particular a leucyl residue. Commonly used synthetic substrates are leucinamide, leucine 4-nitroanilide and leucine hydrazide. This cytosolic zinc-metalloenzyme has been identified in virtually all animal tissues, and most studies have been performed on the enzyme (Mj 324,000) from bovine lens, which has been crystallized. It consists of 6 identical subunits (M, 54,000 487 amino acids 2 Zrf per subunit), and its catalytically active site is in the C-terminal domain. The enzyme is present in many other cells and tissues, e.g. lung, stomach, kidney intestine, serum and leukocytes. In clinical chemistry, this enzyme is a marker for hepatic cell lysis, and may even be a more sensitive marker for acute hepatitis than the aminotransferases. 

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