Spin-labeling studies of urea-treated leucine aminopeptidase.

1. Leucine aminopeptidase (EC 3-4-11-1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-1-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B approximately 4:1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) Mn2+ complexed with the protein is partly released producting hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B approximately 4:1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.     

Influence of glutathione on the catalytic properties of leucine aminopeptidase]

1. Leucine aminopeptidase does not catalyze the hydrolysis of glutathione. 2. Glutathione inhibits the hydrolysis of the substrates leucine hydrazide and leucine-p-nitroanilide by leucine aminopeptidase. 3. By means of kinetic experiments the type of the inhibition has been determined as noncompetitive. The inhibition constant Ki for the Mg2+-activated enzyme is five times higher than for the non-activated enzyme. 4. The degree of inhibition caused by glutathione depends on the pH value indicating a competition between glutathione and OH- ions. Mg2+-activated enzyme is invariably inhibited in the investigated pH range of 7.2 to 9.8. 5. A preincubation of the enzyme with glutathione changes the degree of activity enhancement by metal ions.     

Effect of metal ions on the fluorescence of leucine aminopeptidase and its dansyl-peptide substrates

The effect of Cu(II), Ni(II), Zn(II), Mg(II), and Mn(II) on the fluorescence of porcine kidney cytosol leucine aminopeptidase and three of its dansyl(Dns) peptide substrates, Leu-Gly-NHNH-Dns, Leu-Gly-NH(CH2)2NH-Dns, and Leu-Gly-NH(CH2)6NH-Dns, has been investigated. These five metal ions were chosen for study because each binds to the regulatory metal binding site of leucine aminopeptidase. Since the binding is relatively weak, kinetic studies of the different metalloderivatives of the enzyme are normally carried out in the presence of large molar excesses of these metal ions that can potentially affect both the enzyme and substrate. The fluorescence of all of the dansyl-peptides, as well as several other dansyl species, is quenched by Ni(II) and Cu(II), but not by Mg(II), Mn(II), or Zn(II). The absorption spectra of these dansyl substrates are also perturbed by Ni(II) and Cu(II). The rate at which maximal quenching for some dansyl species is attained after mixing with Ni(II) and Cu(II) is slow and the quenching is reversed on addition of EDTA. These results indicate that the quenching is the result of complex formation between the fluorophores and these metal ions. The association constants for the metal complexes have been determined from Stern-Volmer plots. In addition to complex formation, Ni(II) and Cu(II) cause the degradation of Leu-Gly-NHNH-Dns through a two step mechanism involving loss of dansic acid. Ni(II) and Cu(II) also partially quench the fluorescence of leucine aminopeptidase through contact with its surface accessible Trp residues. These observations indicate that care must be taken in stopped flow fluorescence studies of reactions between this enzyme and its dansyl substrates to avoid adverse effects brought about by Ni(II) and Cu(II).     

Photoinactivation and carbethoxylation of leucine aminopeptidase.

In the present paper the reactivity of histidyl residues of leucine aminopeptidase from bovine eye lens was studied by dye-sensitized photooxidation and by carbethoxylation of the enzyme protein using diethylpyrocarbonate. Of all the different amino acids modified by photooxidation only histidine is connected with the enzymic acticity, whereas tyrosine seems to be involved in structure stabilization. By changing the pH and varying the effectors (Mg2+ and/or dodecylsulfate) of the reaction mixture a different number of histidyl residues of the enzyme protein is caused to react with diethylpyrocarbonate. No secondary reactions with tyrosyl or tryptophyl residues could be observed by spectrophotometric investigations. The enzyme modified by one of the above-mentioned methods shows changes in the capacity of Mn2+ binding measured by autoradiography as well as in the degree of enhancement of enzymic activity by Mn2+ or Mg2+ ions. Of the 48 histidyl residues of the enzyme (Mr = 326000) up to 2 histidyl residues per subunit (Mr = 54000) may be involved in Mn2+ or Mg2+ binding and up to 4 histidyl residues have a strong influence on Zn2+ binding.     

Isolation and properties of leucine aminopeptidase from Aspergillus oryzae]

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.     

Three-dimensional structural resemblance between leucine aminopeptidase and carboxypeptidase A revealed by graph-theoretical techniques.

Using 3-D searching techniques based on algorithms derived from graph theory we have established a striking structural similarity between the structure of bovine carboxypeptidase A and that of the C-terminal domain of bovine leucine aminopeptidase. There is no significant sequence homology between the aminopeptidases and the carboxypeptidases but the strong structural relationship detected in this complex fold suggests that there may be a very remote divergent evolutionary relationship between these two enzyme classes.     

Spectroscopic studies on the formation and thermal stability of DNA triplexes with a benzoannulated delta-carboline-oligonucleotide conjugate

A benzoannulated delta-carboline with a phenyl substituent has been covalently tethered to the 3'-end of a triplex-forming oligonucleotide and its ability to bind and stabilize DNA triple helices has been examined by various spectroscopic methods. UV thermal melting experiments were conducted with different hairpin duplexes and with a complementary single-stranded oligonucleotide as targets for the conjugate. The delta-carboline ligand preferentially binds triplexes over duplexes and leads to a temperature increase of the triplex-to-duplex transition by up to 23 degrees C. The results obtained from UV, CD and fluorescence measurements suggest that the delta-carboline ligand exhibits specific interactions with a triplex and favors binding by intercalation at the triplex-duplex junction.     

Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.     

Spectroscopic studies of (m5dC-dG)3: thermal stability of B- and Z-forms.

The hexanucleoside pentaphosphate d(m5CpGpm5CpGpm5CpG) has been studied in solution by ultra-violet absorption, circular dichroism and 31P nuclear magnetic resonance under various experimental conditions. In 0.2 M NaClO4 at low temperature, an hexamer duplex is formed which has a B or B-like conformation. As the salt concentration is increased, a transition from a B-form to the Z-form occurs and is complete in 3 M NaClO4. In 3 M NaClO4, the behavior of the Z double helix is complex as a function of temperature. The variation of the circular dichroism at 295 nm is biphasic. A first transition occurs over a large range of temperature and corresponds to a conformational change due to a non-cooperative intramolecular process. Ultra-violet absorption and 31P nuclear magnetic resonance show that the new conformation arising from a distortion of the backbone is not similar to that observed in low salt conditions (B-form). At high hexanucleotide concentration, aggregates are formed. The second transition is cooperative and corresponds to the melting of a double stranded helix into single strands.     

Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase.

The pepA gene, encoding a protein with leucine aminopeptidase activity, was isolated from Rickettsia prowazekii, an obligate intracellular parasitic bacterium. Nucleotide sequence analysis revealed an open reading frame of 1,502 bp that would encode a protein of 499 amino acids with a calculated molecular weight of 53,892, a size comparable to that of the protein produced in Escherichia coli minicells containing the rickettsial gene. Also, heat-stable leucine aminopeptidase activity was demonstrable in an E. coli peptidase-deficient strain containing R. prowazekii pepA. Comparison of the amino acid sequence of the R. prowazekii PepA with the characterized leucine aminopeptidases from E. coli, Arabidopsis thaliana, and bovine eye lens revealed that 39.8, 34.9, and 34.0% of the residues were identical, respectively. Residues proposed to be part of the active site or involved in the binding of metal ions in the bovine metalloenzyme were all conserved in R. prowazekii PepA. However, despite the structural and enzymatic similarity to E. coli PepA, the R. prowazekii protein was unable to complement the cer site-specific, PepA-dependent recombination system found in E. coli that resolves ColE1-type plasmid multimers into their monomeric forms.     

Family studies of plasma dopamine-beta-hydroxylase thermal stability.

There are large individual variations in the thermal stability of human plasma dopamine-beta-hydroxylase (DBH). These variations are a characteristic of the DBH molecule itself. Individual subjects may be classified as those with thermolabile and those with thermostable plasma DBH. Of 362 randomly selected unrelated children, 8.01%, and of 238 randomly selected unrelated adult subjects, 5.46% had thermolabile plasma DBH. There was not a significant correlation of DBH thermolability with either sex or age on the basis of data from 230 adults and children in 53 randomly selected families. Subjects with thermolabile DBH had basal enzyme activity only about 55% of that in subjects with stable enzyme. There was not a direct relationship between DBH thermolability and the allele DBHL, the presence of which results in very low basal enzyme activity. There was a significant familial aggregation of the trait of DBH thermolability, but there was not a significant correlation of this trait among spouses. Although preliminary pedigree evaluation raised the possibility of monogenic inheritance of the trait of DBH thermolability by an autosomal recessive mechanism, three separate families in which both parents had thermolabile enzyme included offspring with thermostable DBH. All five of these offspring had very low basal plasma DBH and were presumed to be homozygous for the allele DBHL. These observations raised the possibility that the trait of plasma DBH thermolability may be inherited, and that there may be an interaction between the locus or loci responsible for thermal stability and the locus DBH.     

Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.