Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)_n nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 × 10¹² molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.     

Engineered,highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side‐chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site‐specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100‐fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5‐cd]indolizine‐type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α‐helical, β‐sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q‐GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG‐reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.     

Photolysis and ozonolysis of (iso)desmosine-containing crosslinked peptides from porcine aorta elastin

This report describes the use of photolysis and ozonolysis as a means of achieving complete cleavage of the pyridinium ring of (iso)desmosine in crosslinked elastin peptides. Although photolysis leads to the opening of the ring with concomitant formation of lysine, the peptide chains remain attached. Subsequent ozonolysis is able to completely achieve the cleavage of the rest of the ring skeleton, thus leading to the separation of the peptide chains. Formation of new amino acids, i.e. alpha-aminoadipic and glutamic acids, is emphasized. Localization of these amino acids within the released peptides should be of help in structural investigations on the crosslinking zones involving either isodesmosine or desmosine. However, other amino acids such as tyrosine and phenylalanine are sensitive to this procedure and side reactions occur which are responsible for peptide bond cleavage with the formation of breakdown products.     

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)     

Separation and determination of alpha-amino acids by boroxazolidone formation

Reaction of an alpha-amino acid (alpha-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2-5 in which both the alpha-amino and the alpha-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the greater than N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the alpha-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the alpha-AA moiety of N-terminal gamma-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of alpha-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for alpha-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the epsilon-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an alpha-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze alpha-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from alpha-AA and from those peptides which contain an alpha-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted alpha-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of alpha-amino acids that are derived from post-translational modification of protein side chains.     

Continuous enzyme-coupled assay for microbial transglutaminase activity

Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-ε-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG’s promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K_M = 3 mM) than the commonly used Cbz-Gln-Gly (K_M = 58 mM), attesting to its potential for application in biocatalysis and bioconjugation.     

Basic aspects of the biochemical reactivity of 4-hydroxynonenal

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups. Among the primary reactants for HNE are the amino acids cysteine, histidine and lysine, which--either free or protein-bound--undergo readily Michael additions to the C=C bond. After this primary reaction, which confers rotational freedom to the C2-C3 bond, secondary reactions may occur involving the carbonyl and the hydroxy group. Primary amines may alternatively react with the carbonyl group to form Schiff bases. Reactions which do not fit into this scheme are the oxidation and the reduction respective of the carbonyl group and the epoxidation of the C=C double bond. Examples will be presented for the interaction of HNE with various classes of biomolecules such as proteins and peptides, lipids and nucleic acids and the biochemical consequences will be discussed.     

Synthesis of peptides containing oxo amino acids and their crystallographic analysis

An isolated uncharged hydrogen bond acceptor such as the carbonyl functionality of an aldehyde or a keto group is absent in natural amino acids. Although glutamine and asparagine are known to hydrogen bond through the amide carbonyl group in their side chains, they also possess the amide ? NH₂ group, which can act as a hydrogen bond donor. This makes the structural study of peptides containing an oxo residue, with an isolated carbonyl group in the side chain, interesting. Here, we report the synthesis of δ‐ and ε‐oxo amino acids and their incorporation into oligopeptides as the N‐terminal residue. The resultant oxo peptides were extensively studied using X‐ray crystallography to understand the interactions offered by the oxo group in peptide crystals. We find that the oxo groups are capable of providing additional hydrogen bonding opportunities to the peptides, resulting in increased intermolecular interactions in crystals. The study thus offers avenues for the utilization of oxo residues to introduce intermolecular interactions in synthetic peptides.     

Leucyl/phenylalanyl(L/F)-tRNA-protein transferase-mediated aminoacyl transfer of a nonnatural amino acid to the N-terminus of peptides and proteins and subsequent functionalization by bioorthogonal reactions

We report here a new strategy for derivatizing peptides and proteins at the N-terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N-terminus of a peptide or a protein by using L/F-tRNA-protein transferase. By using the chemoenzymatic technique, beta-(2-quinolyl)-L-alanine, p-azido-L-phenylalanine, and p-acetyl-L-phenylalanine were introduced to the N-terminus. The latter two nonnatural amino acids possess bioorthogonal functional groups to which artificial tags can be introduced. Actually, a biotin tag was coupled to the bioorthogonal ketone group of acetylphenylalanine at the N-terminus of a peptide. N-terminal-specific biotinylation and fluorescence derivatization of the bioorthogonal azido-containing protein or peptide was also carried out based on a [3 + 2] cycloaddition. The enzymatic transfer of a nonnatural amino acid to the N-terminus of target peptides or proteins was also successfully achieved in the presence of other peptides or crude protein mixtures.     

The effect of intercalator structure on binding strength and base-pair specificity in DNA interactions

The interaction of naphthothiophene, phenanthrene and anthracene ring systems, which have amide and ester side chains with cationic groups (synthesized from the aromatic acid chlorides and appropriate amines and alcohols), with calf thymus DNA has been investigated by using viscometric titrations, spectrophotometric binding experiments and 1H-, 31P- and 17O-NMR methods. The viscosity and NMR experiments suggest that all of these compounds bind to DNA by intercalation. These experiments and spectrophotometric binding studies, however, indicate that there is considerable variation in the interaction of these compounds with DNA. These variations can all be explained by the geometry of the ring systems, the position of protons adjacent to the side chains, and the relative sizes of the amide and ester side chains. With the naphthothiophene ester and amide, for example, the planar amide cannot rotate into the plane of the naphthothiophene ring whereas the smaller planar ester can. With this ring system the ester has a significantly higher binding constant than the amide derivative. Additional binding studies with poly[d(A-T)2] and poly[d(G-C)2] have shown that all of these compounds bind more strongly to the A-T- than the G-C-containing polymer. Since the ester compounds do not have hydrogen bond donating groups proximate to the aromatic ring, these results suggest a model for the A-T specificity of these compounds that involves a solvent-mediated hydrogen bond between the C-2 carbonyl of thymine and the carbonyl group of the intercalators.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Structural features facilitating the glutamate dehydrogenase catalyzed alpha-imino acid-alpha-amino acid interconversion

Glutamate dehydrogenase catalyzes the reversible oxidation of L-proline and L-pipecolic acid to the corresponding cyclic alpha-imino acids. The active substrates are the amino acid anion in one direction and the iminium ion in the other. The oxidation of the ester, amide, and N-methyl derivatives of L-proline by enzyme-NADP+ and the reduction of N-methyl-delta 1-tetrahydropyridinium ion by enzyme-NADPH do not proceed to a detectable extent under the experimental conditions. The methyliminium ion, however, undergoes facile nonenzymatic reduction by NADPH. If it is assumed that the nonenzymatic reaction reflects the structural requirements of the redox step of the enzymatic reaction, then the lack of reactivity of the tetrahydropyridinium ion toward enzyme-NADPH must be due to the instability of the central complex. It appears that the alpha-carboxylate and NH groups in the amino acid anion and in the alpha-imino acid are involved in binding the substrates to the enzyme-coenzyme complexes. We conclude that each of these active substrates binds to its appropriate enzyme-coenzyme complex through a hydrogen bond between its NH group and a basic enzyme group; there is also an ionic bond in the central complex between the alpha-carboxylate group of the active substrate and a positively charged enzyme group. The five-membered amino acid anions are more reactive toward enzyme-NADP+ than the six-membered ones. The same reactivity order is seen for the reduction of imino acids by enzyme-NADPH. Since these effects are also present in the nonenzymatic reduction by NADPH we ascribe the ring size effects on V/Ksubstrate primarily to those on the hydride-transfer step.     

Illuminating structure and acyl donor sites of a physiological transglutaminase substrate from Streptomyces mobaraensis

Transglutaminase from Streptomyces mobaraensis (MTG) has become a powerful tool to covalently and highly specifically link functional amines to glutamine donor sites of therapeutic proteins. However, details regarding the mechanism of substrate recognition and interaction of the enzyme with proteinaceous substrates still remain mostly elusive. We have determined the crystal structure of the Streptomyces papain inhibitory protein (SPI_p), a substrate of MTG, to study the influence of various substrate amino acids on positioning glutamine to the active site of MTG. SPI_p exhibits a rigid, thermo‐resistant double‐psi‐beta‐barrel fold that is stabilized by two cysteine bridges. Incorporation of biotin cadaverine identified Gln‐6 as the only amine acceptor site on SPI_p accessible for MTG. Substitution of Lys‐7 demonstrated that small and hydrophobic residues in close proximity to Gln‐6 favor MTG‐mediated modification and are likely to facilitate introduction of the substrate into the front vestibule of MTG. Moreover, exchange of various surface residues of SPI_p for arginine and glutamate/aspartate outside the glutamine donor region influences the efficiency of modification by MTG. These results suggest the occurrence of charged contact areas between MTG and the acyl donor substrates beyond the front vestibule, and pave the way for protein engineering approaches to improve the properties of artificial MTG‐substrates used in biomedical applications.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Metal-ligated amino acids and peptides as substrates for proteases

Amino acids and peptides carrying a pentaamminecobalt(III) group at the carboxyl terminal have been prepared. It is shown that trypsin and papain accept such compounds as substrates provided the metal complex group is not too close to the enzyme-susceptible peptide bond. The possible applicability of this novel type of substrates in enzymatic peptide synthesis is discussed.     

New enzymes for peptide biosynthesis in microorganisms

Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.