Bovine Lens Leucine Aminopeptidase Research Articles

Leucine aminopeptidase: bestatin inhibition and a model for enzyme-catalyzed peptide hydrolysis.

The three-dimensional structures of native bovine lens leucine aminopeptidase (EC 3.4.11.1) and its complex with bestatin, a slow-binding inhibitor, have been solved and exhaustively refined. The mode of binding of bestatin to leucine aminopeptidase may be similar to that of a tetrahedral intermediate that is thought to form during peptide bond hydrolysis. Bestatin binds in the active site with its alpha-amino group and hydroxyl group coordinated to the zinc ion located in the readily exchangeable divalent cation binding site. Its phenylalanyl side chain is stabilized by van der Waals interactions with Met-270, Thr-359, Gly-362, Ala-451, and Met-454, which appear to form a terminal hydrophobic pocket. The leucyl side chain binds in another hydrophobic cleft lined by Asn-330, Ala-333, and Ile-421. Hydrogen bonds involving active site residues Lys-262, Asp-273, Gly-360, and Leu-362 are responsible for stabilizing the backbone nitrogen and oxygen atoms of bestatin. The mode of bestatin inhibition of leucine aminopeptidase is discussed and correlated with biochemical studies of bestatin analogues. In addition, features of a mechanism of catalysis of peptide hydrolysis by leucine aminopeptidase are discussed.

Molecular structure of leucine aminopeptidase at 2.7-A resolution.

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-A resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. The monomers are crystallographically equivalent and each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 A3. The secondary structural composition is 40% alpha-helix and 19% beta-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interactions and does not appear to participate directly in catalysis. The C-terminal domain (327 amino acids) is responsible for catalysis and binds the two zinc ions, which are 2.88 A apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped beta-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-255, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.

The primary structure of leucine aminopeptidase from bovine eye lens.

The amino acid sequence of bovine eye lens leucine aminopeptidase has been determined. Cyanogen bromide fragments, the COOH-terminal hydroxylamine fragment, and a large fragment obtained by digestion with Staphylococcus aureus protease were isolated from reduced and S-alkylated leucine aminopeptidase. The amino acid sequences of these fragments were determined by automated sequence analysis, by manual direct Edman degradation, and by the dansyl-Edman technique. Overlapping peptides were obtained by tryptic digestion of the S-alkylated protein or the citraconylated S-alkylated protein. The polypeptide chain of leucine aminopeptidase comprises 478 residues, corresponding to a molecular weight of 51,691. No significant sequence homology with any other published protein primary structure could be detected. This is the first report of a complete amino acid sequence of an enzyme belonging to the class of two metal peptidases.

Sulfhydryl content of bovine eye lens leucine aminopeptidase. Determination of the reactivity of the sulfhydryl groups of the zinc metalloenzyme, of the enzyme activated by Mg2+, Mn2+, and Co2+, and of the metal-free apoenzyme.

The reactivity of the sulfhydryl groups of leucine aminopeptidase from bovine eye lens has been studied by carboxymethylation with iodoacetate of the native enzyme (Zn2+-Zn2+), of the enzyme activated with magnesium (Zn+-Mg2+) or manganese (Zn2+-Mn2+), the enzyme incubated with cobalt (CO2+-Co2+), and the metal-free apoenzyme. The reactivity of the sulfhydryl groups was also determined in the presence of denaturing agents with or without reducing agents. All seven half-cystines per leucine aminopeptidase subunit are in the sulfhydryl form. In the native and the Zn2+-Mn2+ enzyme, only one of the cysteines (residue 344) reacts readily with iodoacetate. In the Zn2+-Mg2+ enzyme, two cysteines react (residues 344 and 412) and in the Co2+-incubated enzyme only one (residue 412). The metal-free apoenzyme has at least three reactive cysteines (residues 344, 412, and 429). The data suggest that sulfhydryl groups are involved in the binding of metal ions.

Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins.

Rabbit antisera to bovine lens leucine aminopeptidase (LAP) have been prepared. These LAP-specific antisera cross-react with a component, LAP, in human lens homogenates. Bovine and human lens LAP are similar but not identical. Immunodiffusion tests show that LAP is present in a vast majority if not all cataractous and normal human lens homogenates. Results from immunoelectrophoresis indicate that LAP is found in these homogenates as several metazymes as well as in an albuminoid--and possibly membrane-associated form. In contrast to many activity-based studies which imply that very little LAP is present in human lenses, micro-complement fixation tests indicate that the concentration of LAP in aged human lenses is similar to that found in calf lenses. Taken together, these data indicate that LAP undergoes age-related changes. These alterations of enzyme (or environment) result in an enzyme of markedly reduced activity, hence, the discrepancy between amount of LAP and the amount of enzymatic activity in aged human lenses. Active LAP is shown to enhance the rate of hydrolysis of alpha-2 and alpha-B crystallins but not alpha-A2, beta H, beta L or gamma crystallins. The ramifications of LAP inactivation with respect to cataractogenesis are discussed.

The primary structure of bovine lens leucine aminopeptidase: Complete amino acid sequence of the N-terminal cyanogen bromide fragment and site of limited tryptic digestion

The amino acid sequence of the N-terminal cyanogen bromide fragment of bovine lens leucine aminopeptidase has been determined. This fragment contains a total of 171 amino acid residues and has a calculated molecular weight of 18,637. The sequence data presented here represent the first report of primary structure determination of a member of the class of aminopeptidases. The single cleavage site produced by limited tryptic digestion of native leucine aminopeptidase was determined to be between arginine-137 and lysine-138 of the total amino acid sequence. The possible existence of distinct structural domains in leucine aminopeptidase is discussed.

Limited tryptic digestion of bovine eye lens leucine aminopeptidase

Limited tryptic digestion of bovine eye lens leucine aminopeptidase

Leucine Aminopeptidase from Bovine Eye Lens

Specific modification of bovine eye lens leucine aminopeptidase by the substrate-like and radioactive tagged diazonium peptide inhibitor, L-(2,3-3H)Phe(pN2+)-L-Phe, was investigated using the technic of affinity and differential labelling. The degree of labelling and the loss of activity increased with excess of the label. A competitive inhibitor diminished the degree of azo-coupling and also the degree of inactivation. First results of tryptic digestion of labelled LAP are given.

Kinetic Properties of Bovine Lens Leucine Aminopeptidase

Kinetic studies with bovine eye lens leucine aminopeptidase are reported. Particular attention is paid to the substrate specificity, the transferase activity, the correlation between binding and catal

Preliminary X-ray study of leucine aminopeptidase (bovine lens), an oligomeric metalloenzyme

Bovine lens leucine aminopeptidase (EC no. 3.4.1.1) crystallizes in the hexagonal space group P6322 with unit cell dimensions a = 132 Å and c = 122 Å. The asymmetric unit consists of one protomer of molecular weight 54,000. The 32 point group symmetry of the hexameric molecule establishes the presence of two types of isologous interactions between dyad-related subunits.

The suitability of bovine-lens leucine aminopeptidase for sequence analysis and limited hydrolysis of polypeptides.

In the present paper we report findings showing the suitability of recrystallized bovine‐lens leucine aminopeptidase as a tool for peptide chemistry.The enzyme is homogeneous with respect to hydrodynamic, chromatographic and electrophoretic separation criteria. It is not contaminated by endopeptidases.The insulin B‐chain (S‐sulfonate) served as model substrate for sequence studies. The initial progress curves of the liberation of the first six amino acids resulted in a sequence expected from the known primary structure. The complete resistance of imido bonds towards the action of leucine aminopeptidase has been proved.Many commercially available protein preparations are found to be contaminated by endopeptidases. We developed an endopeptidase‐detection method in which the leucine aminopeptidase serves as a sensitivity amplifier.

Bovine-lens leucine aminopeptidase. Kinetic studies with activators and competitive inhibitors.

An attempt has been made, by means of kinetic experiments, to get some insight into the molecular mechanism of the binding and catalytic steps taking place during low‐molecular‐weight substrate hydrolysis by leucine aminopeptidase from bovine eye lenses. To this end the variation of the kinetic parameters with concentration and type of well‐known activators (anions and metal cations of valency two) was studied systematically.Additional, we searched for substrate‐like competitive inhibitors. It was found that the end product l‐leucine shows no competition but the compounds N‐p‐tosyl‐N′‐butylcarbamide and N‐sulfanilyl‐N′‐butylcarbamide turned out to be rather potent competitive inhibitors with Ki values comparable to the Km of l‐leucine‐p‐nitroanilide. The variation of the ki values with temperature was studied so that the thermodynamic parameters of inhibitor binding could be calculated.In order to be able to make a meaningful comparison between the kinetic parameters all data were subjected to a computer‐aided (CDC 1604‐A) statistical analysis.