Kinetics of amino acid esterification catalyzed by hydrophobic derivatives of alpha-chymotrypsin

Hexyl-alpha-chymotrypsin, a hydrophobic derivative of the enzyme, is explored for the proteinase-catalyzed ester synthesis reaction with N-acetyl-L-tyrosine and ethanol. To guarantee the preservation of the enzyme activity and to allow for the extraction of the product in the organic phase, a biphasic system was used. The Vm increased for the modified enzyme. This phenomenon was linked to the modification of the protein as demonstrated by its chemical denaturation with sodium dodecyl sulfate.     

Transfection activity of polyamidoamine dendrimers having hydrophobic amino acid residues in the periphery

We designed poly(amidoamine) dendrimers with phenylalanine or leucine residues at their chain ends. Thereby, we achieved efficient gene transfection of cells through synergy of the proton sponge effect, which is induced by the internal tertiary amines of the dendrimer, and hydrophobic interaction by the hydrophobic amino acid residues in the dendrimer periphery. Dendrimers having 16, 29, 46, and 64 terminal phenylalanine residues were prepared by the reaction of the amine-terminated poly(amidoamine) G4 dendrimer and L-phenylalanine using condensing reagent 1,3-dicyclohexylcarbodiimide. Transfection activity of these phenylalanine-modified dendrimers for CV1 cells, an African green monkey kidney cell line, increased concomitant with the increasing number of the terminal phenylalanine residues, except for the dendrimer with 64 phenylalanine residues, which showed poor water solubility and hardly formed a complex with DNA at neutral pH. However, under weakly acidic conditions, the dendrimer with 64 phenylalanine residues formed a complex with DNA, thereby achieving highly efficient transfection. In contrast, the attachment of L-leucine residues was unable to improve the transfection activity of the parent dendrimer, probably because of the relatively lower hydrophobicity of this amino acid. The phenylalanine-modified dendrimer exhibited a higher transfection activity and a lower cytotoxicity than some widely used transfection reagents. For that reason, the phenylalanine-modified dendrimers are considered to be promising gene carriers.     

Completion of the amino acid sequence of papain

Papain was inhibited with bromo[2-(14)C]acetic acid, the tertiary structure of the inhibited enzyme was unfolded and the disulphide bridges were reduced with mercaptoethanol and aminoethylated. Digestion with trypsin gave a radioactive peptide consisting of residues 18-58 inclusive and containing therefore the sequence of the thirteen unknown residues 29-41 in the primary sequence of papain. This peptide was digested with pepsin to give a radioactive peptide consisting of residues 18-47, which after digestion with 0.4m-hydrochloric acid gave a radioactive peptide consisting of residues 24-43 inclusive. Further digestion with 6m-hydrochloric acid gave peptides that were used to determine the sequence: Ser-Ala-Val-Val-Thr-Ile-Glx-Gly-Ile-Ile-Lys-Ile-Arg for the residues 29-41, so completing the amino acid sequence of papain.     

Glycine flanked by hydrophobic bulky amino acid residues as minimal sequence for effective subtilisin catalysis.

The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1' residue and predilection for bulky hydrophobic P2' residues. A synergism in the action of S1' and S2'subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.     

Fast bilayer-micelle fusion mediated by hydrophobic dipeptides

To understand the transition from inanimate matter to life, we studied a process that directly couples simple metabolism to evolution via natural selection, demonstrated experimentally by Adamala and Szostak. In this process, dipeptides synthesized inside precursors of cells promote absorption of fatty acid micelles to vesicles, inducing their preferential growth and division at the expense of other vesicles. The process is explained on the basis of coarse-grained molecular dynamics simulations, each extending for tens of microseconds, carried out to model fusion between a micelle and a membrane, both made of fatty acids in the absence and presence of hydrophobic dipeptides. In all systems with dipeptides, but not in their absence, fusion events were observed. They involve the formation of a stalk made by hydrophobic chains from the micelle and the membrane, similar to that postulated for vesicle-vesicle fusion. The emergence of a stalk is facilitated by transient clusters of dipeptides, side chains of which form hydrophobic patches at the membrane surface. Committor probability calculations indicate that the size of a patch is a suitable reaction coordinate and allows for identifying the transition state for fusion. Free-energy barrier to fusion is greatly reduced in the presence of dipeptides to only 4–5 kcal/mol, depending on the hydrophobicity of side chains. The mechanism of mediated fusion, which is expected to apply to other small peptides and hydrophobic molecules, provides a robust means by which a nascent metabolism can confer evolutionary advantage to precursors of cells.     

Enantioselective hydrolysis of hydrophobic amino acid derivatives by lipases

Microbial lipases preferentially cleave the L-isomers of N-benzyloxycarbonyl (Z)-hydrophobic D,L-amino acid methyl esters which is the same as that of subtilisin. This implies that lipases and proteases may share the same ancestor in the evolutionary point and lipases can be practically applied to resolve racemic hydrophobic amino acid derivatives.     

Separation of some glycyl dipeptides with the amino acid analyzer

The chromatographic behavior of 7 glycyl dipeptides under standard conditions of automatic amino acid analysis in 30–50° and 50° systems has been examined. We separated these peptides in 18 and 10.5 hr, respectively, and found that they never overlapped with the peak position of the constituent amino acids.     

Identification of hydrophobic amino acid residues involved in the formation of S100P homodimers in vivo

S100 proteins are small dimeric members of the EF-hand superfamily of Ca(2+) binding proteins thought to participate in mediating intracellular Ca(2+) signals by binding to and thereby regulating target proteins in a Ca(2+)-dependent manner. As dimer formation is crucial to S100 function, we applied a yeast two-hybrid approach in analyzing in vivo molecular aspects of S100 dimerization. We chose S100P, a member of the S100 family highly expressed in placenta, for detailed analysis and showed that S100P monomers strongly interact with one another but not with other S100 polypeptides, indicating that homodimer formation is obligatory for S100P. Analysis of the interaction of site-specific S100P mutants with the wild-type polypeptide or with other S100P mutant chains identifies conserved hydrophobic amino acid residues involved in mediating dimerization in vivo. Of these residues, F-15 is crucially important as a mutation to alanine abolishes dimerization even when the F15A S100P mutant polypeptide is allowed to interact with a wild-type chain. On the other hand, I-11, I-12, or F-89 need to be replaced by a less hydrophopic residue in both subunits for there to be a similar extent of interfere with dimerization. This proves that hydrophobic residues implicated through structural studies in S100 dimerization are involved in the dimer interaction in vivo and argues for a hierarchy of hydrophobic contacts stabilizing the dimer and thereby regulating S100 function.     

A functional prolipoprotein signal peptide with a deletion of four amino acid residues from the hydrophobic region

The deletion of several codons within the signal sequence coding region of the Escherichia coli lipoprotein gene has been accomplished by oligonucleotide-directed site-specific mutagenesis. The deletion of the Leu-13 residue in a mutant in which two glycine residues had previously been deleted from the hydrophobic region (Inouye, S., Vlasuk, G., Hsiung, H., and Inouye, M. (1984) J. Biol. Chem. 259, 3729-3733) was found to cause the accumulation of the unmodified form of the protein in the cytoplasm and cytoplasmic membrane. This mutation also caused a cessation in cell growth within 15 min after synthesis of the mutant protein was induced. A deletion of the Val-7 residue was capable of suppressing the effect of the Leu-13 deletion when both are present. However, by itself the Val-7 deletion appeared to have little effect on the glycine mutant. The ability of the signal sequence to mediate the secretion of the protein after the deletion of 4 residues from the hydrophobic region demonstrates a surprising degree of flexibility in the length of this region. The deletion mutations were also found to have an unusual effect on the rate of synthesis of lipoprotein.     

The structural code for proteins: zonal distribution of amino acid residues and stabilization of helices by hydrophobic triplets

The general distribution of 2259 amino acid residues in α-helical and in non-helical regions has been calculated using data from 12 proteins. The irregular distribution of hydrophobic residues in the helical parts of the sample permits classification of the helical regions in hydrophobic-clustered and hydrophobic-depleted zones. In comparison with the composition of α-helices, Ala, Leu, Val, Ile and Tyr predominate in hydrophobic-clustered zones, whereas Cys and Gln tend to accumulate in hydrophobic-depleted zones. The N-terminal part of the helices is rich in Asp and Pro and poor in Leu and His, whereas the C-terminal is rich in Lys and Gin and does not contain Pro.For helical regions, hydrophobic residues, located along the sequence with a relation of 1-2-5 or 1-4-5, are more frequent than those with a relation of 1-2-3, 1-2-4, 1-3-4 or 1-3-5. The opposite is found for non-helical regions. All these deviations are statistically significant. It is concluded that hydrophobic triplets 1-2-5 and 1-4-5 are required for stabilizing the helices.The relevance of our results, and others reported in the bibliography, towards a better understanding of the structural code for proteins is discussed.     

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.     

疏水性氨基酸的羟基化研究进展

羟基化氨基酸在生物技术和分子生物学中具有独特价值,具有抗真菌、抗菌、抗病毒和抗癌的特性。通过比较化学合成与生物催化合成羟基氨基酸的异同,选择具有高对映结构选择性的生物催化合成方法成为羟基氨基酸合成的首选。生物催化实现疏水性氨基酸的羟基化和羟化酶紧密相关,而羟化酶又是单核非血红素Fe(Ⅱ)和α-酮戊二酸依赖型双加氧酶(Fe/αKGDs)的一种,Fe/αKGDs存在共性催化机制。因此,疏水性氨基酸在被催化的过程中,会利用关键中间体高价铁-超氧复合体(Fe(Ⅳ)=O)引起多种氧化转化,从而完成羟基化过程。文中就疏水性氨基酸的羟基化合成及功能应用,尤其是(2S,3R,4S)-4-羟基-异亮氨酸(4-HIL)和羟脯氨酸,进行了详细的阐述,探讨了Fe/αKGDs的共性催化反应机制,并对羟基氨基酸在基础研究和工业中的应用进行了综述。     

A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly

Neuronal and oligodendrocytic aggregates of fibrillar alpha-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise viability of affected cells. Here, we report that a 12-amino acid stretch ((71)VTGVTAVAQKTV(82)) in the middle of the hydrophobic domain of human alpha-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta-synuclein is highly homologous to alpha-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha-synuclein filaments, because it is resistant to proteolytic digestion in alpha-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha-synuclein fibrillization and might arrest disease progression.     

Demetallation and esterification reactions of heterocyclic amino acid complexes

The copper(II) complex of 4-methyloxazolidine- 4′-carboxylic acid and the nickel(II) complex of 3N,7N-(1,3,5,7-tetraazabicyclo [3.3.1]nonyl)diacetic acid were prepared and then treated with sodium borohydride to form the sodium salts of the respective acids. The saturated heterocyclic rings of the acids are retained in the reactions.The methyl esters of the acids were subsequently prepared under anhydrous conditions and characterized by gas chromatography/mass spectrometry (GC/MS).     

Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues

Patterns of hydrophobic and hydrophilic residues play a major role in protein folding and function. Long, predominantly hydrophobic strings of 20-22 amino acids each are associated with transmembrane helices and have been used to identify such sequences. Much less attention has been paid to hydrophobic sequences within globular proteins. In prior work on computer simulations of the competition between on-pathway folding and off-pathway aggregate formation, we found that long sequences of consecutive hydrophobic residues promoted aggregation within the model, even controlling for overall hydrophobic content. We report here on an analysis of the frequencies of different lengths of contiguous blocks of hydrophobic residues in a database of amino acid sequences of proteins of known structure. Sequences of three or more consecutive hydrophobic residues are found to be significantly less common in actual globular proteins than would be predicted if residues were selected independently. The result may reflect selection against long blocks of hydrophobic residues within globular proteins relative to what would be expected if residue hydrophobicities were independent of those of nearby residues in the sequence.     

Enzyme inhibition by dipeptides containing 2,3-methanophenylalanine, a sterically constrained amino acid

Both isomers of (E)-2,3-methanophenylalanine (^EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)- and (2S,3R)-^EPhe-Phe(or Leu)-OMe were found to inhibit effectively the hydrolysis of Ac-Tyr-OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the ^EPhe-Phe peptide bond was also entirely stable. The inhibition constant (K_i) of the most potent dipeptide of H-(2R,3S)-^EPhe-Phe-OMe was 0.16 mM at 25°C. The inhibitory action of Phe-containing peptides was found to depend on the configuration of the Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H-(2R,3S)-^EPhe-Leu-OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.     

Protease-catalyzed esterification of amino acid in water-miscible ionic liquid

The activity of two proteases in the esterification of N-acetyl-L-phenylalanine with ethanol was examined in the water-miscible ionic liquid, 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([emim][Tf]). The activity of subtilisin was not only improved 9-fold by changing from a water-miscible organic solvent, acetonitrile, to [emim][Tf], but also was about three times greater than that in a water-immiscible organic solvent, octane. Likewise, the activity of alpha-chymotrypsin in [emim][Tf] was more effectively enhanced compared with that in a water-miscible or a water-immiscible organic solvent. The water content in [emim][Tf] affected the activity of subtilisin.