Investigation on the substrate specificity of human plasmin using tripeptidyl-p-nitroanilide substrates

The hydrolysis of 35 tripeptidyl-p-nitroanilides was studied with human plasmin and the kinetic parameters were determined. The individual contribution of the various side chains to the kinetic parameters was calculated by regression analysis. Considering Km, substrates having Z-D-Ile-Phe-Lys as well as H-D-Ile-Phe-Lys sequences were found to be the best, while Bz-Ile-Leu-Lys and pGlu-Leu-Lys sequences are the best for kcat. The Km values of substrates protected at N-terminus are lower, their kcat values are higher than those of the unprotected ones with the same sequence.     

Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases

A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.     

Yeast dipeptidase: active site mapping by kinetic studies with substrates and substrate analogs.

In order to characterize the active site of yeast dipeptidase in more detail, kinetic studies with a variety of dipeptide substrates and substrate analogs were performed. To analyze kinetic data, computer programs were developed which first calculate initial velocities from progress curves and then evaluate the kinetic parameters by nonlinear regression analysis. A free carboxyl group is a prerequisite for binding of dipeptidase substrates; its position relative to the peptide bond must not deviate from the normal L-dipeptide conformation. The spatial arrangement of the terminal ammonium ion seems to be less crucial. The enzyme's substrate specificity clearly reflects the interactions of the substrate amino acid side chains with complementary dipeptidase subsites. The domain of the enzyme in contact with the C-terminal substrate side chain seems to be an open structure of moderately hydrophobic character. In contrast, the binding site for the amino-terminal side chain is a more strongly hydrophobic "pocket" of limited dimensions. The kinetics of inhibition by free amino acids points to an ordered release of products from the enzyme.     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

Affinity chromatography of subtilisin on a sorbent with epsilon-aminocapronyl-alanyl-alanyl-D-leucylamide. Detection of a serine protease with unusual properties]

A biospecific sorbent obtained by attachment of epsilon-aminocapronyl-L-alanyl-L-alanyl-D-leucylamide to CNBr-activated Sepharose 4B has been used for affinity chromatography of various samples of subtilisin BPN', e.g. subtilisin A ("Serva"), Nagarse, A-50. Two active components were isolated from subtilisin A (Serva"), the major component corresponding to subtilisin BPN' and the minor component (SII) being a serine proteinase with low molecular weight (about 10000). The molecular weight and amino acid composition of SII as well as the kinetic parameters of its action on peptide substrates (p-nitroanilides of N-benzyloxycarbonyl-Gly-Gly-Leu, -Ala-Ala-Leu, -Gly-Gly-Phe, -Ala-Ala-Phe. The low molecular weight proteinase possesses a high affinity for the leucine residue in P1 position and alanine in P2 and P3 positions. The specificity of this proteinase differs from that of the main component.     

Kinetics of glycogen phosphorylase a with a series of semisynthetic, branched saccharides. A model for binding of polysaccharide substrates.

The requirement of muscle phosphorylase for branched polysaccharide substrates was investigated by kinetic studies on semisynthetic branched saccharides. One series of saccharides was prepared from maltoheptose by oxidizing the reducing group to a carboxyl group and coupling this with an amino group of ethylenediamine. The resulting aminooligosaccharide was coupled with p-nitrophenyl esters of mono-, di-, tetra-, and polycarboxylic aicds to produce saccharides containing one, two, four, and approximately 52 maltodextrin chains per molecule. A similar series of saccharides was prepared from a heterogeneous maltodextrin of average chain length 11.7. Kinetic constants were determined for the reaction with phoshorylase a in the direction of chain elongation. Michaelis constants are equilibrium constants for dissociation of saccharide from the enzyme-AMP-glucose-1P-saccharide complex. The Michaelis constants, expressed in terms of the concentration of nonreducing end groups, are independent of maltodextrin chain length but decrease considerably as the number of chains per molecule increases. Maximum velocities do not differ greatly from that for glycogen. Among the synthetic saccharides, only the polymer behaves similarly to glycogen in exhiiting a decreasing reaction rate as the chains are elongated. The kinetic constants are quantitatively consistent with a model in which two chain termini from the same saccharide molecule bind to the phosphorylase molecule simultaniously, Differences in binding between saccharides having different numbers of equally accessible chains are caused solely by statistical factors in the equilibrium. Highly branched substrates bind better because of their greater multiplicity of two end-group pairs.     

Optimal spatial requirements for the location of basic residues in peptide substrates for the cyclic AMP-dependent protein kinase

The specificity of the cyclic AMP-dependent protein kinase was examined using two series of dodecapeptides as substrates. One series consisted of peptides of the general sequence (Gly)x-Arg-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y = 6. The other series consisted of peptides of the sequence (Gly)x-Lys-Arg-(Gly)y-Ala-Ser-Leu-Gly in which x + y was again equal to 6. The peptides Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Arg-Ser-Leu-Gly and Gly-Gly-Gly-Gly-Gly-Gly-Gly-Lys-Arg-Ser-Leu-Gly were also examined. In the series in which the adjacent arginines were located various distances from the serine, the substrate for which the enzyme clearly exhibited optimal kinetic constants contained one amino acid residue between the basic residues and serine. Direct binding studies of N alpha-[3H]acetyl peptides to catalytic subunit of cyclic AMP-dependent protein kinase revealed a correlation between binding affinity and the ability to serve as substrate for the enzyme. In the second series in which the adjacent basic amino acids were Lys-Arg, optimal kinetic constants were again obtained when these residues were separated from serine by a single amino acid. This latter result was surprising in view of phosphorylation site sequences in the known physiologically significant protein substrates for the kinase, since those containing Lys-Arg all contain two amino acids between these residues and serine.     

Molecular design of fluorescent labeled glycosides as acceptor substrates for sialyltransferases

A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α(1)-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V(max)/K(m)) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K(m) value for α2,6- and α2,3-sialyltransferases.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Effect of P2' substituents on kinetic constants for hydrolysis by cysteine proteinases.

Intramolecularly quenched fluorogenic peptide substrates with the general sequence: DABCYL-Lys-Phe-Gly-Gly-Xxx-Ala-EDANS have been utilized to explore the effect of the hydrophobicity of amino acid side chains in the P2' position on the steady-state kinetic constants for papain catalyzed hydrolysis. The results demonstrate that subsite interactions between the enzyme and the peptide substrate modulate the enzyme specificity by slowing the release of the C-terminal product. This series of substrates can be used to characterize substrate specificity studies of other cysteine proteinases.     

The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents

To investigate how the conformational flexibility of subtilisin affects its ability to discriminate between enantiomeric amino acid and ester substrates for the subtilisin-catalyzed reaction in an organic solvent, the flexibility around the active site and the surface of subtilisin was estimated from the mobility of a spin label bound to subtilisin by ESR spectroscopy. Many studies on enzyme flexibility focus on the active site. Both the surface and active site flexibility play an important role in the enantioselectivity enhancement of the enzyme-catalyzed reaction. It was found, however, that the different behavior observed for the enantioselectivity between the amino acid and ester substrates could be correlated with the flexibility around the surface rather than the flexibility at the active site of subtilisin. In other words, for the ester substrates, the greater flexibility around the surface of subtilisin induced by a conformational change resulting from the presence of an additive such as DMSO is essential for the enantioselectivity enhancement. This model is also supported by the Michaelis-Menten kinetic parameters for each enantiomeric substrate. Our findings provide insight into the enantioselectivity enhancement for the resolution of enantiomers for enzyme-catalyzed reactions in organic solvents.     

Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin

The preparations of N alpha-Fmoc-3-nitro-L-tyrosine and N-Boc-anthranilic acid Dhbt ester and their application to parallel multiple column solid-phase peptide synthesis is described. A series of peptide substrates containing an anthraniloyl group at the amino terminus and a 3-nitrotyrosyl residue close to the carboxyl terminus have been synthesized. The fluorescence of the anthraniloyl group, intramolecularly quenched by the 3-nitrotyrosine, increases with cleavage of peptide bonds situated between the two groups. The quenching mechanism is of the long-range resonance energy transfer type and long peptide substrates were constructed and used for kinetic measurement on subtilisin Carlsberg and pepsin. Complete quenching was observed even with more than 20 A between the centers of the chromophores, and substrates with up to 50 A between the chromophores were synthesized. The importance of long substrates for optimal enzymatic activity was demonstrated.     

Synthesis of plasmin substrates and relationship between their structure and plasmin activity

The plasmin substrates, D-Ile-Phe-Lys-pNA (I), 3-MV-Phe-Lys-pNA (II), Ile-Phe-Lys-pNA (III), D-Pro-Phe-Lys-pNA (IV), CP-Phe-Lys-pNA (V) and Pro-Phe-Lys-pNA (VI), were synthesized by the conventional solution method and the kinetic parameters of their amidolysis by plasmin were determined. It was found that the free amino group of the D-amino acid in substrates (I) and (IV) made a contribution to an increment in affinity between the substrate and plasmin.     

N-anthraniloyl-Ala-Ala-Phe-4-nitroanilide, a highly sensitive substrate for subtilisins.

A new substrate for subtilisins, anthraniloyl-Ala-Ala-Phe-4-nitroanilide, has been synthesized and characterized. The peptide is a fluorogenic substrate that is intramolecularly quenched without loss of its chromogenic properties and offers a possibility for double-assay kinetic analysis. The kinetic parameters determined for subtilisin Carlsberg are Km = 0.004 mM, kcat = 104 s-1, and those for subtilisin BPN' are Km = 0.020 mM, kcat = 49 s-1. The substrate is extremely sensitive for subtilisins; the specificity constants are 10-fold higher than the corresponding values for the widely used substrate, succinyl-Ala-Ala-Pro-Phe-4-nitroanilide, and 200- to 1000-fold higher than the values obtained with succinyl-Ala-Ala-Phe-4-nitroanilide. The favorable effect of the anthraniloyl group as a P4 residue in the substrate sequence Ala-Ala-Phe-4-nitroanilide was assumed to be due to an ability to stiffen S4-P4 interactions. The mechanism proposed is hydrogen bond formation between the phenol group of tyrosine-104 and the amino group of the anthraniloyl moiety. In the spectrophotometric assay with the new substrate, the lower detection limit for subtilisin Carlsberg was 1 nM.     

Isolation and characterization of novel tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.     

Substrate specificity of the human protein phosphatase 2Cdelta, Wip1

Wip1, the wild-type p53-induced phosphatase, selectively dephosphorylates a threonine residue on p38 MAPK and mediates a negative feedback loop of the p38 MAPK-p53 signaling pathway. To identify the substrate specificity of Wip1, we prepared a recombinant human Wip1 catalytic domain (rWip1) and measured kinetic parameters for phosphopeptides containing the dephosphorylation sites in p38alpha and in a new substrate, UNG2. rWip1 showed properties that were comparable to those of PP2Calpha or full-length Wip1 in terms of affinity for Mg(2+), insensitivity to okadaic acid, and threonine dephosphorylation. The substrate specificity constant k(cat)/K(m) for a diphosphorylated peptide with a pTXpY sequence was 6-8-fold higher than that of a monophosphorylated peptide with a pTXY sequence, while PP2Calpha showed a preference for monophosphorylated peptides. Although individual side chains before and after the pTXpY sequence of the substrate did not have a significant effect on rWip1 activity, a chain length of at least five residues, including the pTXpY sequence, was important for substrate recognition by rWip1. Moreover, the X residue in the pTXpY sequence affected affinity for rWip1 and correlated with selectivity for MAPKs. These findings suggest that substrate recognition by Wip1 is centered toward a very narrow region around the pTXpY sequence. Three-dimension homology models of Wip1 with bound substrate peptides were constructed, and site-directed mutagenesis was performed to confirm the importance of specific residues for substrate recognition. The results of our study should be useful for predicting new physiological substrates and for designing specific Wip1 inhibitors.     

Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.     

Influence of enzyme-substrate contacts located outside the EcoRI recognition site on cleavage of duplex oligodeoxyribonucleotide substrates by EcoRI endonuclease.

A complete understanding of the sequence-specific interaction between the EcoRI restriction endonuclease and its DNA substrate requires identification of all contacts between the enzyme and substrate, and evaluation of their significance. We have searched for possible contacts adjacent to the recognition site, GAATTC, by using a series of substrates with differing lengths of flanking sequence. Each substrate is a duplex of non-self-complementary oligodeoxyribonucleotides in which the recognition site is flanked by six base pairs on one side and from zero to three base pairs on the other. Steady-state kinetic values were determined for the cleavage of each strand of these duplexes. A series of substrates in which the length of flanking sequence was varied on both sides of the hexamer was also examined. The enzyme cleaved both strands of each of the substrates. Decreasing the flanking sequence to fewer than three base pairs on one side of the recognition site induced an asymmetry in the rates of cleavage of the two strands. The scissile bond nearest the shortening sequence was hydrolyzed with increasing rapidity as base pairs were successively removed. Taken together, the KM and kcat values obtained may be interpreted to indicate the relative importance of several likely enzyme-substrate contacts located outside the canonical hexameric recognition site.     

New substrates for enteropeptidase. I. Biologically active hepta-nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue provided that less than four negatively charged amino acid residues are in positions P2-P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR decreases VYIHPF) and the Hb(2-8) (LTAEEK decreases A) and Hb(1-9) (MLTAEEK decreases AA) peptides of the cattle hemoglobin beta-chain. The Km values for all the substrates (approximately 10(-3) M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the -DDDDK- full-size linker (Km approximately 10(-4) M). The kcat values for AT and Hb(2-8) were also close and low (approximately 30 min-1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2-8) peptide by one amino acid residue from its N- or C-terminus results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1-9) is 1510 min-1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Role of charge and hydrophobic effects in reactions of peptide substrates and inhibitors with thrombin

A substrate and inhibitor analysis of the thrombin interaction with synthetic peptide substrates and inhibitors of differing hydrophobicity and volume of the side amino acid residue, localized in the sub-centers thrombin S2 and S3 were carried out. The kinetic parameters of individual stages of the enzymatic reaction process (K(S), k2, k3) were estimated. It is shown that the efficiency of acylation and deacylation stages of the enzymatic reaction decreases with increasing hydrophobicity of the substituent in P2 as well as P3, at the same time the affinity of selected peptides toward enzyme is steadily increasing. With the aim to evaluate the hydrophobicity of compounds a LogP value was calculated and was made an attempt to compare them with the correspondent Ki values. Comparative kinetic analysis of Z-Arg-OMe and its uncharged analogue Z-Cit-OMe has shown the absence of uncharged analog hydrolysis, however, the mentioned citrulline derivate inhibits the hydrolysis of the charged analogue. These findings confirm the important role of hydrophobic moiety in the structure of thrombin inhibitors in preferential binding mode and inhibition of thrombin active side.