Isolation and characterization of mammalian D-aspartyl endopeptidase

Summary. The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer’s disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH₂Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC₅₀, 3 μM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.     

Mammalian D-aspartyl endopeptidase: a scavenger for noxious racemized proteins in aging

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease, cataracts, and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600kDa) and is localized in the inner mitochondrial membrane of mouse and rabbit, but DAEP activity was not detected in Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans. A specific inhibitor for DAEP was newly synthesized, and inhibited DAEP activity (IC(50), 3microM), a factor of 10 greater than lactacystin on DAEP. On the other hand, the inhibitor did not inhibit either the 20S or 26S proteasome.     

Paenidase, a novel D-aspartyl endopeptidase from Paenibacillus sp. B38: purification and substrate specificity

We discovered and characterized a novel type D-aspartyl endopeptidase (DAEP) produced extracellularly by Paenibacillus sp. B38. This bacterial DAEP of M(r) 34,798 (named paenidase) appeared to be converted into a smaller form of M(r) 34,169 by the proteolytic removal of 5 amino acid residues from the N-terminal. The intact and modified forms of the enzyme displayed essentially the same enzymatic properties. The enzyme specifically hydrolyzed succinyl-D-aspartic acid alpha-(p-nitroanilide) and succinyl-D-aspartic acid alpha-(4-methylcoumaryl-7-amide) to generate p-nitroaniline and 7-amino-4-methylcoumarin, and internally cleaved a synthetic peptide (D-A-E-F-R-H-[D-Asp]-G-S-Y) of the [D-Asp](7) amyloid beta (Abeta) protein between [D-Asp](7)-G(8). Either was totally inert to the normal Abeta peptide sequence containing L-Asp, instead of D-Asp at the 7th position. Thus, paenidase is the first DAEP from a microorganism that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds.     

Substrate specificity and stereoselectivity of hydrolytic enzymes from Brevibacterium imperiale B222

Four different hysrolytic enzymes were isolated and partially purified from Brevibacterium imperiale B222 cells. The stereoselectivity of each enzyme was assayed by using the nitrile, amide and esters derivatives of 2-aryloxypropionic acid. Within the cellular pool of hydrolytic enzymes, a non-stereoselective nitrile hydratase, a stereoselective amidase and two partially stereoselective esterases of opposite enantiomeric preference were found.Correspondence to: D. Bianchi     

Substrate specificity and stereoselectivity of fatty alcohol oxidase from the yeast Candida maltosa

Summary In Candida maltosa and other alkene-utilizing yeasts a membrane-bound fatty alcohol oxidase (FAOD) is induced by growth on n-alkenes. The oxidation of 1-alkanols to the corresponding aldehydes is accompanied by the stoichiometric consumption of 1 mol O₂ and formation of 1 mol hydrogen peroxide (H₂O₂). The FAOD of C. maltosa shows a broad substrate specificity. It catalyses the oxidation of 1-alkanols (C₄ to C₂₂), with a maximal activity of 1.85 gmmol H₂O₂/ min × mg protein for 1-octanol, as well as the transformation of 2-alkanols (C₈ to C₁₆) to ketones. Other compounds as ,-alkenediols, -hydroxypalmitic acid, phenylalkanols and terpene alcohols are substrates for the enzyme, although mostly with decreased activities. The oxidation of the racemic 2-alkanols by the FAOD proceeds with very high stereoselectivity for the R(–)-enatiomers only, leaving the S(+)-2-alkanol untouched. Offprint requests to: S. Mauersberger     

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.     

Expression of enzymatically active enkephalinase (neutral endopeptidase) in mammalian cells

A cDNA encoding the rat enkephalinase protein (neutral endopeptidase; EC 3.4.24.11) has been constructed from overlapping lambda gt10 cDNA clones. This cDNA was inserted into an expression plasmid containing the cytomegalovirus enhancer and promoter. When transfected with this plasmid, Cos 7 cells transiently expressed the enkephalinase protein in a membrane-bound state. Recombinant enkephalinase recovered in solubilized extracts from transfected Cos 7 cells was enzymatically active and displayed properties similar to those of the native enzyme with respect to sensitivity to classical enkephalinase inhibitors.     

Substrate phosphorylation affects degradation and interaction to endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme

Recent findings from our laboratory suggest that intracellular peptides containing putative post-translational modification sites (i.e., phosphorylation) could regulate specific protein interactions. Here, we extend our previous observations showing that peptide phosphorylation changes the kinetic parameters of structurally related endopeptidase EP24.15 (EC 3.4.24.15), neurolysin (EC 3.4.24.16), and angiotensin-converting enzyme (EC 3.4.15.1). Phosphorylation of peptides that are degraded by these enzymes leads to reduced degradation, whereas phosphorylation of peptides that interacted as competitive inhibitors of these enzymes alters only the K(i)'s. These data suggest that substrate phosphorylation could be one of the mechanisms whereby some intracellular peptides would escape degradation and could be regulating protein interactions within cells.     

Substrate specificity of HeLa endonuclease R. A G-specific mammalian endonuclease

We examined the substrate specificity of endonuclease R (endo R) a mammalian endonuclease that cleaves G.C-rich DNA sequences. The best substrates for double-stranded cleavage were homopolymeric stretches of poly(dG).poly(dC). Plasmids which contain other G-rich sequences were also cleaved but at a reduced frequency. These included the telomeric sequences, d(G4T2) and d(G2-6A), which were cleaved at approximately one-third the frequency of d(G)n.d(C)n. The alternating copolymer d(GA) and the terminal sequences of adeno-associated virus d(G1-3T/A) were also cut. Poly(dA).poly(dT) and the alternating copolymer d(GC)n were not detectably cleaved. Although endo R has a nicking activity which converts supercoiled plasmids to nicked circular DNA, the nicking activity is random with respect to plasmid sequences. Specific cleavage of G-rich sequences appears to occur by a concerted double-stranded mechanism. The cleavage pattern within the G-rich runs suggests that cleavage can occur anywhere within the G-rich region. Product ligation experiments indicate that a limited number of cleavage events (1-2) occur/molecule. Inasmuch as the best substrates for endo R are d(G)n.d(C)n and telomeric sequences, we suggest that endo R may directly recognize and cleave DNA that contains G.G base pairing.     

Understanding lipase stereoselectivity

Microorganisms or isolated enzymes can be applied as catalysts to create highly regio- and stereoselective conversions under mild conditions. Lipases (EC 3.1.1.3, triacylglycerol lipase) are lipid-hydrolysing enzymes, which are increasingly used in stereoselective reactions. Their industrial importance arises from the fact that they act on a variety of substrates promoting a broad range of biocatalytic reactions. Lipase stereoselectivity is exploited for the production of single enantiomers instead of racemic mixtures and will become more important in the pharmaceutical and agrochemical industry because, in most cases only one of the two enantiomers has the desired activity, whereas no activity or even undesirable side effects reside in the other enantiomer. Enantiomer differentiation is due to the various diastereomeric interactions that occur between the enantiomers and the active site of the enzyme. The stereospecificity of a lipase depends largely on the structure of the substrate, interaction at the active site and on the reaction conditions. Stereoselectivity involves a wide range of factors such as differentiation of enantiotopes, differentiation of enantiomers, type of substrate, biochemical interaction of the substrate with the enzyme, steric interaction of the substrates, competition between two different substrates, nature and availability of the active site for stereoselective action, presence of water and nature of solvents based on polarity and supercritical state. This article reviews factors responsible for lipase stereoselectivity.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using Xenopus laevis oocytes as an expression system and by combining the two-electrode voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-α-amino acids substrates can consist maximally of only one CH₂-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH₂-units, as in γ-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cysteine only in the form of the D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analysis of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations that determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and side chain size, whereas substitutions on the amino group are well tolerated.     

Prolyl endopeptidase

Prolyl endopeptidase (E.C. 3.4.21.26) an enzyme previously called post proline cleaving enzyme, TRH-deamidase or kininase B, may play a role in neuropeptide metabolism. This enzyme, highly active in brain and other tissues, catabolizes proline-containing peptides such as substance P, neurotensin, luteinizing hormonereleasing hormone, thyrotropin releasing hormone, bradykinin and angiotensin II. The structure of β-neo-endorphin suggests that this opioid peptide is formed by the action of prolyl endopeptidase on a precursor of higher molecular weight. Formation of two biologically active fragments of substance P also requires the action of this enzyme. This review summarizes the current knowledge of the biochemistry of this enzyme, and its potential significance for neuropeptide physiology and pharmacology.