Lens aminopeptidase

The alkaline phosphatase family contains three metals in the active site while the rest of the cocatalytic zinc sites contain two metals (Figme 10) (Table 3). Some of these sites contain metals such as copper, iron, and magnesium in combination with zinc. Combinations of Cu(II)/Zn are seen in the superoxide dismutase (SOD) family see Copper Proteins with Type 2 Sites), Zn/Mg are seen in alkaline phosphatase family and lens aminopeptidase, and Fe(III)/Zn in the purple acid phosphatase family. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

In spite of the large eflFects of or on the activity of lens aminopeptidase (and probably on the kidney enzyme as well), it is doubtful if they play any physiological role. The relative concentrations of Zn ", Mg and Mn " and the pH of the particular tissues in question are such as to preclude formation of any hybrid metalloenzymes in vivo. However, the fact that the activation does occur indicates its potential for occurring in vivo although it may not be brought about by Mg " or Mn2 ... 

It was clear for some time that a number of zinc enzymes required two or more metal ions for full activity, but in the absence of X-ray structural data the location of these metal centres with regard to one another was often uncertain. When the first 3-D structures began to appear, it became clear that the metals were in close proximity. A particular feature of many of these enzymes was the presence of a bridging ligand between two of the metal sites, usually an Asp residue of the protein, which is occasionally replaced by a water molecule. While some of the sites contain only Zn ions, several contain Zn in combination with Cu (in cytosolic superoxide dismutases) Fe (in purple acid phosphatases) or Mg (in alkaline phosphatase and the aminopeptidase of lens). 

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... 

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... 

A Taylor, MJ Brown, MA Daims, J Cohen (1983) Localization of leucine aminopeptidase in normal hog lens by immunofluorescence and activity assays, Invest Ophthalmol Vis Sci 24 1172-1180... 

A Taylor, T Surgenor, DK Thomson, RJ Graham, H Oettgen (1984) Comparison of leucine aminopeptidase from human lens, beef lens and kidney, and hog lens and kidney, Exp Eye Res 38 217-229... 

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. 

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

Aminopeptidases are present in many tissues (Table III). Leucine aminopeptidase from intestinal mucosa is very effective in catalyzing the hydrolysis of leucine from the amino terminus of peptides, polypeptides, and proteins. It also hydrolyzes leucine amide and leucine esters (10). The designation leucine aminopeptidase is somewhat of a misnomer because activity is also observed when other amino acids replace leucine. Only the L-isomers of amino acids are substrates, and the presence of a D-amino acid residue or proline in the penultimate position will retard hydrolysis (10). Enzymes having the same specificity as the intestinal aminopeptidase have been identified and/or isolated from kidney, pancreas, muscle, lens, and various bacterial sources (10). The kidney... 

Some of the more extensive studies have been with the aminopeptidase from bovine lens (18, 19). Though this enzyme is not involved in protein digestion, some of its characteristics are presented here since they may be closely analogous to those of the enzyme from intestinal mucosa. 

The first evidence indicating that leucine aminopeptidase is a metallo-enzyme was obtained only recently (20, 21) although metals have long been known to aflfect its activity (22). Both the lens and the kidney... 

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. 

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... 

Aminopeptidase Family bovine lens IBLL Znl 2.9 GlUaa... 

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,... 

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...

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. 

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]. 

The oligopeptide may also be sequenced from the AT-terminus by an enzymic method using the cytosol aminopeptidase of pig kidney or cattle lens. This enzyme acts on all peptide bonds except arginyl or lysyl bonds. The concentrations of identified free amino acids are measured at given time intervals. The amino acid sequence of the... 

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

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