Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Oxidized elafin and trappin poorly inhibit the elastolytic activity of neutrophil elastase and proteinase 3

Neutrophil proteinase-mediated lung tissue destruction is prevented by inhibitors, including elafin and its precursor, trappin. We wanted to establish whether neutrophil-derived oxidants might impair the inhibitory function of these molecules. Myeloperoxidase/H(2)O(2) and N-chlorosuccinimide oxidation of the inhibitors was checked by mass spectrometry and enzymatic methods. Oxidation significantly lowers the affinities of the two inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This decrease in affinity is essentially caused by an increase in the rate of inhibitory complex dissociation. Oxidized elafin and trappin have, however, reasonable affinities for NE (K(i) = 4.0-9.2 x 10(-9) M) and for Pr3 (K(i) = 2.5-5.0 x 10(-8) M). These affinities are theoretically sufficient to allow the oxidized inhibitors to form tight binding complexes with NE and Pr3 in lung secretions where their physiological concentrations are in the micromolar range. Yet, they are unable to efficiently inhibit the elastolytic activity of the two enzymes. At their physiological concentration, fully oxidized elafin and trappin do not inhibit more than 30% of an equimolar concentration of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin strongly impairs their activity. Inhibitor-based therapy of inflammatory lung diseases must be carried out using oxidation-resistant variants of these molecules.     

Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching.

Subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites. Interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes. The development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the active sites of subtilisin BPN' from Bacillus amyloliquefaciens and Savinase from Bacillus lentus. For each enzyme, the eight positions S5-S'3 were characterized by determination of kcat/KM values for the hydrolysis of substrates in which the amino acids were systematically varied. The results emphasize that in both subtilisin BPN' and Savinase interactions between substrate and S4 and S1 are very important. However, it is apparent that interactions between other subsites and the substrate exert a significant influence on the substrate preference. The results are rationalized on the basis of the structural data available for the two enzymes.     

Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5

Mannan-binding lectin-associated serine proteases (MASPs) are secreted as single-chain precursors and processed into two disulfide bond-linked chains. MASP-3 and MASP-1, derived from the same gene, contain identical A chains, but entirely different catalytic domain-containing B chains. In contrast to MASP-1 and MASP-2, the proteinase activity of MASP-3 has not been described previously. We show here the proteolytic activity of the purified recombinant human MASP-3 catalytic domain toward peptides and protein substrates. Among the fluorogenic peptides tested, it specifically cleaved peptides with Arg at the P1 position. Among seven insulin-like growth factor-binding proteins, it selectively cleaved IGFBP-5, which is the first protein substrate identified for MASP-3. All three cleavage sites identified contained Arg or Lys at the P1 position and Pro at the P2 position. As compared to MASP-1 and MASP-2, MASP-3 has distinct substrate specificity and inhibitor profile. These results should be useful for further studies of the structure and function of human MASP-3.     

Influence of charge distribution at the active site surface on the substrate specificity of human neutrophil protease 3 and elastase. A kinetic and molecular modeling analysis

The biological functions of human neutrophil protease 3 (Pr3) differ from those of neutrophil elastase despite their close structural and functional resemblance. Although both proteases are strongly cationic, their sequences differ mainly in the distribution of charged residues. We have used these differences in electrostatic surface potential in the vicinity of their active site to produce fluorescence resonance energy transfer (FRET) peptide substrates for investigating individual Pr3 subsites. The specificities of subsites S5 to S3' were investigated both kinetically and by molecular dynamic simulations. Subsites S2, S1', and S2' were the main definers of Pr3 specificity. Combinations of results for each subsite were used to deduce a consensus sequence that was complementary to the extended Pr3 active site and was not recognized by elastase. Similar sequences were identified in natural protein substrates such as NFkappaB and p21 that are specifically cleaved by Pr3. FRET peptides derived from these natural sequences were specifically hydrolyzed by Pr3 with specificity constants k(cat)/K(m) in the 10(6) m(-1) s(-1) range. The consensus Pr3 sequence may also be used to predict cleavage sites within putative protein targets like the proform of interleukin-18, or to develop specific Pr3 peptide-derived inhibitors, because none is available for further studies on the physiopathological function of this protease.     

Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.     

The substrate specificity of SARS coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome coronavirus (SARS) has been proposed to be a key target for structural based drug design against SARS. We have designed and synthesized 34 peptide substrates and determined their hydrolysis activities. The conserved core sequence of the native cleavage site is optimized for high hydrolysis activity. Residues at position P4, P3, and P3' are critical for substrate recognition and binding, and increment of beta-sheet conformation tendency is also helpful. A comparative molecular field analysis (CoMFA) model was constructed. Based on the mutation data and CoMFA model, a multiply mutated octapeptide S24 was designed for higher activity. The experimentally determined hydrolysis activity of S24 is the highest in all designed substrates and is close to that predicted by CoMFA. These results offer helpful information for the research on the mechanism of substrate recognition of coronavirus 3C-like proteinase.     

Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition

The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, Ac-VDVAD-Cho and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and Ac-VDVAD-Cho have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of Ac-VDVAD-Cho, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase.     

Triple-helical peptide analysis of collagenolytic protease activity

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 > MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.     

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.     

Activity profiling of human deSUMOylating enzymes (SENPs) with synthetic substrates suggests an unexpected specificity of two newly characterized members of the family

SENPs [Sentrin/SUMO (small ubiquitin-related modifier)-specific proteases] include proteases that activate the precursors of SUMOs, or deconjugate SUMOs attached to target proteins. SENPs are usually assayed on protein substrates, and for the first time we demonstrate that synthetic substrates can be convenient tools in determining activity and specificity of these proteases. We synthesized a group of short synthetic peptide fluorogenic molecules based on the cleavage site within SUMOs. We demonstrate the activity of human SENP1, 2, 5, 6, 7 and 8 on these substrates. A parallel positional scanning approach using a fluorogenic tetrapeptide library established preferences of SENPs in the P3 and P4 positions that allowed us to design optimal peptidyl reporter substrates. We show that the specificity of SENP1, 2, 5 and 8 on the optimal peptidyl substrates matches their natural protein substrates, and that the presence of the SUMO domain enhances catalysis by 2-3 orders of magnitude. We also show that SENP6 and 7 have an unexpected specificity that distinguishes them from other members of the family, implying that, in contrast to previous predictions, their natural substrate(s) may not be SUMO conjugates.     

Cleavage of bovine collagen I by neutrophil collagenase MMP-8. Effect of pH on the catalytic properties as compared to synthetic substrates

The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37 degrees C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of (1/4) and (3/4) fragments. Further, the first cleavage event has been investigated at 37 degrees C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k(cat)/K(m), k(cat), and K(m). These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK(a) values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates.     

Role of the S1' subsite glutamine 215 in activity and specificity of stromelysin-3 by site-directed mutagenesis.

The influence of Gln215 in stromelysin-3 (MMP-11), a residue located in the S1' subsite, was determined by producing three single mutants of this position. As compared to wild-type stromelysin-3, the kinetic parameters K(M) and k(cat) for the degradation of the fluorogenic substrate Dns-Pro-Leu-Ala-Leu-Trp-Ala-Arg-NH(2) (Dns-Leu) by these mutants indicated that the Gln/Leu substitution led to a 4-fold decrease in catalytic efficiency, whereas the mutations Gln/Tyr and Gln/Arg increased this parameter by a factor 10. The cleavage of alpha1-protease inhibitor (alpha1-PI), a natural substrate of stromelysin-3, by these mutants was also determined. Their relative activities for the degradation of alpha1-PI correspond to those observed with the synthetic substrate Dns-Leu. The catalytic efficiency of wild-type stromelysin-3 and its mutants to cleave the P1' analogue of Dns-Leu, containing the unusual amino acid Cys(OMeBn) (Dns-Cys(OMeBn)), was also determined. The values of the specificity factor, calculated as the ratio (k(cat)/K(M))Dns-Cys(OMeBn))/(k(cat)/K(M))Dns-Leu, were observed to vary from 26 for the wild-type stromelysin-3 to 120 for the Gln/Leu mutant and 25 for the Gln/Arg mutant. The Gln/Tyr mutant did not cleave the substrate when its P1' position is substituted by the unusual amino acid Cys(OMeBn). Altogether these observations established that both the catalytic activity and the specificity of stromelysin-3 are dependent on the nature of the residue in position 215. Finally, the cleavage efficiency of the Dns substrates by three representative matrixins, namely, MMP-14 (215 = Leu), MMP-1 (215 = Arg), and MMP-7 (215 = Tyr), was determined. Interestingly, the trends observed for these enzymes were similar to those established for the three mutants of stromelysin-3, pointing out the influence of position 215 toward the selectivity in this family of enzymes.     

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.     

Biochemical analysis of the 20 S proteasome of Trypanosoma brucei

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.     

Multicatalytic, High-Mr Endopeptidase from Postmortem Human Brain

The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.     

Effect of GTP analogues on purified soluble guanylate cyclase

In our studies with purified soluble guanylate cyclase from rat lung, we have tested a number of guanosine 5'-triphosphate (GTP) analogues as substrates and inhibitors, 5'-Guanylylimidodiphosphate (GMP-P(NH)P), guanylyl (beta, gamma-methylene) diphosphate (GMP-P(CH2)P), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were found to be substrates for guanylate cyclase. GTP gamma S supported cyclic GMP formation at 20 or 75% of the rate seen with Mn2+-GTP and Mg2+-GTP, respectively. GMP-P(NH)P and GMP P(CH2)P supported cyclic GMP formation at 10-20% of the GTP rate with either cation cofactor. These analogues were found to have multiple Km values; one Km value was similar to GTP (150 microM with Mg2+, 20-70 microM with Mn2+), but an additional high affinity catalytic site (3 microM) was also observed. Guanosine tetraphosphate (Ki = 10 microM), adenosine triphosphate (Ki = 9 microM) and the 2'3'-dialdehyde derivative of GTP (dial GTP) (Ki = 1 microM) were not good substrates for the enzyme; however, they were potent competitive inhibitors. These GTP analogues will be useful tools for the study of GTP binding sites on guanylate cyclase and they may also help elucidate the effects of free radicals and other agents on guanylate cyclase regulation.     

Arabinose 5-phosphate analogues as mechanistic probes for Neisseria meningitidis 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the condensation reaction between phosphoenolpyruvate and D-arabinose 5-phosphate (D-A5P) in a key step in lipopolysaccharide biosynthesis in Gram-negative bacteria. The KDO8P synthase from Neisseria meningitidis was cloned into Escherichia coli, overexpressed and purified. A variety of D-A5P stereoisomers were tested as substrates, of these only D-A5P and l-X5P were substrates. The Asn59Ala mutant of N. meningitidis KDO8P synthase was constructed and this mutant retained less than 1% of the wild-type activity. These results are consistent with a catalytic mechanism for this enzyme in which the C2 and C3 hydroxyl groups of D-A5P and Asn59 are critical.     

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.     

Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P(5) and P(5)' positions, respectively. In addition, Arg was incorporated in the P(2)' and P(8)' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly approximately Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K(M) = 61.2 microM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta(243-450))], and a C-terminal domain-deleted MMP-3 [MMP-3(Delta(248-460))]. The order of k(cat)/K(M) values was MMP-13 > MMP-1 approximately MMP-1(Delta(243-450)) approximately MMP-2 > MMP-3 approximately MMP-3(Delta(248-460)). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.