Partial intercalation with DNA of peptides containing two aromatic amino acids

The interactions with DNA of tetrapeptide amides containing lysine at the N-terminal position and aromatic amino acids at the second and fourth positions (Ala at position three), 1-6, have been investigated by nmr, CD, and viscometric methods. Tetrapeptides with N-terminal lysine and a single aromatic amino acid, 7-10, were investigated as controls. Significant decreases in DNA viscosity occurred on addition of 7, with the aromatic group at the second position, but not with any of the other single aromatic amino acid peptides. All of the tetrapeptides with two aromatic groups caused DNA viscosity decreases which were two to three times larger than with 7. Peptides with p-nitrophenylalanine (p-NO2Phe) as the aromatic group were synthesized for nmr studies because of its simpler aromatic nmr spectrum relative to Phe. Large upfield shifts of the aromatic proton signals were obtained when the amino acid in the second position was L-p-NO2Phe, and the fourth position contained either p-NO2Phe or Phe. Such peptides also caused the largest DNA viscosity decreases on complex formation. Smaller upfield shifts of the aromatic signals were obtained when the amino acid in the second position was L-Phe or a D isomer of Phe or p-NO2Phe. With all peptides, larger upfield nmr shifts were obtained with heat-denatured, recooled DNA than with native DNA under the same conditions. As with nmr, CD results are quite different for the peptides with L and D amino acids at the second position. All of the results can be interpreted in terms of a model in which lysine interacts stereospecifically with the backbone in a DNA double helix and the aromatic group at the second position stacks strongly with the base pairs when the amino acid is an L isomer. The aromatic group at the fourth position can also interact with the base pairs, but primarily through a sideways stacking of the aromatic group with base pairs for either L or D isomers. Because of covalent constraints on the separation distance for the two aromatic groups in the tetrapeptides, they must stack on opposite sides of the same base pair in violation of the neighbor exclusion principle observed with classical intercalators. This stacking at the same base pair no doubt accounts for the larger viscosity decreases in DNA with the peptides containing two aromatic groups relative to those with a single aromatic group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues.

We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.     

Electrostatically driven immobilization of peptides onto (Maleic anhydride-alt-methyl vinyl ether) copolymers in aqueous media

The covalent immobilization of a model peptide onto the MAMVE copolymer, via the formation of amide bonds, occurred in moderate yields in aqueous conditions. The improvement of the grafting reaction was achieved by adding at the amino terminus of the model peptide a sequence (tag) of three positively charged amino acids, lysine or arginine, and by taking profit of electrostatic attractive interactions between the negatively charged copolymer and the tagged peptides. The arginine tag was more efficient than the lysine tag for enhancing the immobilization reaction, proving that the effect was due to an electrostic driving force. On the basis of these results, a tentative mechanism is discussed, and Scatchard plots pointed out two regimes of binding. With the first, at low polymer load (up to 50% of saturation for a lysine tag and 60-70% for an arginine tag), the binding occurred with a positive cooperative effect, the already bound peptide participating to the binding of others. A second one for higher coverages, for which the binding occurred with a negative cooperativity, and saturation was reached in the presence of a large excess of peptide.     

Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis

The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

Endothelium dependent vascular relaxation by arginine containing polypeptides

The vascular endothelium appears obligatory for the expression of the vasodilating property of most polypeptides. A number of polypeptides were studied on the rat aortic ring preparation which was pre-contracted with phenylephrine and only basic polypeptides containing one or more arginine residues elicited relaxation which was endothelium dependent. These peptides included melittin and poly-L-Arg. The basic polypeptide poly-L-Lys also elicited endothelium dependent relaxation, but to a lesser extent than arginine containing polypeptides. Two basic polypeptides, apamin and mastoparan do not promote endothelium dependent relaxation. The former contains arginine between disulfide bonds and in the latter arginine is absent. Basic amino acids and dipeptides which contain arginine, and also polyamines did not elicit relaxation even at high concentrations (10(-3) M). The relaxation elicited by melittin, poly-L-Arg and poly-L-Lys was inhibited by ETYA, NDGA, p-bromophenacyl bromide and not by indomethacin. Methylene blue, an inhibitor of soluble guanylate cyclase, also abolished the relaxation. We suggest that arginine containing peptides may relax vascular smooth muscle by acting directly on the vascular smooth muscle (eg: atriopeptins) and/or or by eliciting release of a relaxing factor(s) from the endothelium.     

Effects of synthetic model peptides resembling the extension peptides of mitochondrial enzyme precursors on import of the precursors into mitochondria

One common and characteristic feature of the extension peptides of mitochondrial enzyme precursors is the presence of repeating short stretches of uncharged amino acids linked by basic amino acids. We synthesized several model peptides having this particular feature of the extension peptides. The peptides contained arginine or lysine as a basic amino acid residue linking sequences of two to four residues of leucine and alanine. We examined the effects of the peptides on the import of the precursors of two mitochondrial enzymes, cytochrome P-450(SCC) and adrenodoxin, and found that the peptides were generally inhibitory to the import of the precursors into mitochondria. The effective concentrations of some of the inhibitory peptides were as low as a few microM. The peptides containing lysine instead of arginine had an essentially similar inhibitory effect on the import. The peptides did not inhibit the binding of pre-P450(SCC) to the surface of mitochondria. The synthetic model peptides uncoupled oxidative phosphorylation of mitochondria prepared from either rat liver or bovine adrenal cortex, and induced leakage of enzymes from the inner compartments of mitochondria. However, the synthetic model peptides did not solubilize membrane-bound enzymes from mitochondria, suggesting that their effect on the membranes is different from that of detergents. The synthetic model peptides seem to bind to the membranes causing significant perturbation in the membrane structure, which is possibly related to the functions of the particular common sequence found in the extension peptides of mitochondrial enzyme precursors.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Identification of formaldehyde-induced modifications in proteins: reactions with model peptides

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.     

Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.

Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.     

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.     

The Presence of Arginine in the Pro-Arg-Pro Motif Augments the Lethality of Proline Rich Antimicrobial Peptides of Insect Source

Antimicrobial peptides are emerging as alternate drug candidates over couple of decades. The diversity exists in amino acid sequence, conformation and mechanism of action of these peptides. Cationic antimicrobial peptides constitute major class which is further classified depending on abundance of amino acid. Insect originated cationic proline rich antimicrobial peptides do not destabilize bacterial cell membrane but have intracellular targets. Some of the peptides belonging to proline rich class such as Apidaecins and Drosocin have unique tripeptide, Pro-Arg-Pro motif which is absent in Formaecin I of same group. Earlier we have designed a non-glycosylated analog of Formaecin I which contains a Pro-Lys-Pro motif. In this report, we have shown that substitution of lysine to arginine in Pro-Lys-Pro motif increases lethal action of this peptide against all the tested bacterial strains without affecting its structural, cytotoxic and membrane permeabilization properties. Importance of arginine in Pro-Arg-Pro motif is reemphasized when substitution of arginine to lysine in Pro-Arg-Pro, tripeptide sequence of Apidaecin and Drosocin results into decrease in their activity. Maintaining overall charge, this substitution indicates the role of arginine beyond providing cationicity.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.     

Sites of phosphorylation on the high molecular weight basic nuclear proteins of the winter flounder

The high molecular weight basic nuclear proteins from winter flounder sperm are a group of at least 16 proteins which are on average 1000 amino acids long. Despite their large size and complexity, this group of proteins gave rise to only 20 major peptides when digested with trypsin, suggesting that they are closely related to each other and contain sequences which are repeated internally many times over. When the phosphorylated forms of the high molecular weight basic nuclear proteins were isolated from mid-spermatid nuclei and digested with trypsin, six of the major peptides were shown to be phosphorylated. Five of these phosphopeptides were purified, and sequenced by automated Edman degradation. Each of the five contained all or part of the consensus sequence X-Ser(P)-X-Ser(P)-Pro, where X represents lysine or arginine. These phosphorylation sites which may be repeated an average of 20 times in each of the high molecular weight proteins, are different from the phosphorylation sites seen in other basic nuclear proteins. The dephosphorylated high molecular weight basic nuclear proteins from flounder sperm are phosphorylated in vitro at these sites by mid-spermatid nuclei.     

More monensin-sensitive, ammonia-producing bacteria from the rumen

Two monensin-sensitive bacteria which utilized carbohydrates poorly and grew rapidly on amino acids were isolated from the bovine rumen. The short rods (strain SR) fermented arginine, serine, lysine, glutamine, and threonine rapidly (greater than 158 nmol/mg of protein per h) and grew faster on casein digest containing short peptides than on free amino acids ().34 versus 0.29 h(-1)). Gelatin hydrolysate, an amino acid source containing an abundance of long peptides, was unable to support growth or ammonia production, but there was a large increase in ammonia production if strain SR was cocultured with peptidase-producing ruminal bacteria (Bacteroides ruminicola or Streptococcus bovis). Cocultures showed no synergism with short peptides. Strain SR washed out of continuous culture ().1 h(-1)) at pH 5.9. The irregularly shaped organisms (strain F) deaminated glutamine, histidine, glutamate, and serine rapidly (greater than 137 nmol/mg of protein per min) and grew faster on free amino acids than on short peptides ().43 versus 0.21 h(-1)). When strain F was provided with casein or gelatin hydrolysate and cocultured with peptidase-producing bacteria, there was a more than additive increase in ammonia production. Strain F grew in continuous culture (0.1 h(-1)) when the pH was as low as 5.3. The irregularly shaped cells and short rods were present at less than 10(9)/ml in vivo, but they ahd very high specific activities of ammonia production (greater than 310 nmol of ammonia/mg of protein per min) and could play an important role in ruminal amino acid fermentation.     

Molecular structure of L-lysyl-L-tyrosyl-L-serine acetate

The structure of the tripeptide L-lysyl-L-tyrosyl-L-serine acetate was determined by X-ray diffraction. The crystals are triclinic space group P1, with two peptide molecules in the unit cell. The peptides are in zwitterionic form with positive charges both in the amino terminal and epsilon-amino groups of lysine. A negative charge is found in one of the carboxylic groups, whereas the other one is protonated. Both peptides show very similar backbone torsional angles, in the beta pleated sheet region, but different tyrosine and serine side-chain conformations. The two lysine side chains have a similar conformation g + tg + t, which had not been previously found. In the unit cell we also find one water molecule, one isopropanol molecule and four acetic acid molecules, three of them likely to be present as acetate anions. These molecules form layers which separate the beta-pleated sheets. The whole structure looks like an ordered solution of peptides in the beta-sheet conformation. An extensive network of hydrogen bonds stabilizes the crystal structure.     

A Novel Type of Substrate Specificity of Rice BAPAase (Benzoyl-L-arginine p-nitroanilide Hydrolase) with Mixed Endopeptidase and Carboxypeptidase

The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984)     

Analysis of recognition elements for mitochondrial processing peptidase using artificial amino acids: roles of the intervening portion and proximal arginine.

We recently demonstrated, using synthetic peptides modeled on the extension peptide of malate dehydrogenase, that amino acid residues present at the proximal and distal positions relative to the cleavage site are critical determinants for the recognition of substrates by mitochondrial processing peptidase [Niidome et al. (1994) J. Biol. Chem. 269, 24719-24722). While the proximal arginine is unexceptionally located at the -2 position, the position of the distal residue varies among mitochondrial precursor proteins. Between the proximal and distal residues, proline and/or glycine are present in most mitochondrial precursor proteins, and they are considered to play a role in the specific recognition of a substrate by the peptidase. To elucidate the role of the intervening portion, we introduced a non-natural amino acid [2-(2-aminoethoxy)acetic acid] between the distal and proximal residues. We also analyzed the functional elements in the proximal arginine by replacing the residue with various arginine or lysine analogs. The results of kinetic studies indicated that the intervening portion should be flexible for efficient processing, and that the guanidino group of the proximal arginine is recognized by the peptidase through hydrogen and ionic bonds.