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.     

Structural Insights into the Catalytic Domains of Human Matrix Metalloprotease-2 and Human Matrix Metalloprotease-9: Implications for Substrate Specificities

Structural information for the gelatinases A (MMP-2) and B (MMP-9), two members of the matrix metalloprotease (MMP) family of enzymes, has been elusive. For the first time, computational structures for the catalytic domains of MMP-2 and MMP-9 are reported herein using the program COMPOSER and the reported three-dimensional structures of the fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and stromelysin-1 (MMP-3). The details of the structures of the catalytic domains of gelatinases and interactions with the protein substrate are discussed. The first analysis of the extent of hydrophobicity of surfaces in the active sites of six MMPs (including the two gelatinases reported herein) is presented to provide distinction for substrate specificity among these metalloproteases. The information from the extent of hydrophobicity/hydrophilicity analysis and general topology for these MMPs was utilized in the proposal of a method for categorization of MMPs of known three-dimensional fold. These efforts provide the first information useful to experimentalists working on the biochemical properties of these important members of the MMP family of enzymes, and provide for an opportunity to compare and contrast structures of gelatinases, collagenases and stromelysins.Electronic Supplementary Material available.     

Structural Insights into the Catalytic Domains of Human Matrix Metalloprotease-2 and Human Matrix Metalloprotease-9: Implications for Substrate Specificities

Structural information for the gelatinases A (MMP-2) and B (MMP-9), two members of the matrix metalloprotease (MMP) family of enzymes, has been elusive. For the first time, computational structures for the catalytic domains of MMP-2 and MMP-9 are reported herein using the program COMPOSER and the reported three-dimensional structures of the fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8) and stromelysin-1 (MMP-3). The details of the structures of the catalytic domains of gelatinases and interactions with the protein substrate are discussed. The first analysis of the extent of hydrophobicity of surfaces in the active sites of six MMPs (including the two gelatinases reported herein) is presented to provide distinction for substrate specificity among these metalloproteases. The information from the extent of hydrophobicity/hydrophilicity analysis and general topology for these MMPs was utilized in the proposal of a method for categorization of MMPs of known three-dimensional fold. These efforts provide the first information useful to experimentalists working on the biochemical properties of these important members of the MMP family of enzymes, and provide for an opportunity to compare and contrast structures of gelatinases, collagenases and stromelysins.     

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.     

Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity. Suggestion of a common topological specificity for functionally diverse proteases

Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.     

Exosite interactions impact matrix metalloproteinase collagen specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.     

Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD(+)-dependent M2DH was confirmed. The enzyme is a monomer of 58kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0-10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting (k(cat)/K) or saturating (k(cat)). The pH-dependence of logk(cat) was usually different from that of log(k(cat)/K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log(k(cat)/K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.     

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.     

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.     

Oligopeptidase B: a new type of serine peptidase with a unique substrate-dependent temperature sensitivity

Oligopeptidase B, a member of the novel prolyl oligopeptidase family of serine peptidases, is involved in cell invasion by trypanosomes. The kinetic analysis of the reactions of oligopeptidase B, which preferentially cleaves peptides at two adjacent basic residues, has revealed significant differences from the trypsin-like serine peptidases. (i) The pH dependence of k(cat)/K(m) deviates from normal bell-shaped curves due to ionization of an enzymatic group characterized by a macroscopic pK(a) of approximately 8.3. The effect of this group is abolished at high ionic strength. (ii) The second-order acylation rate constants, k(cat)/K(m), are similar with the ester and the corresponding amide substrates, suggesting that their chemical reactivity does not prevail in the rate-limiting step. The kinetic deuterium isotope effects indicate that the rate-limiting step for k(cat)/K(m) is principally governed by conformational changes. (iii) The pH-k(cat)/K(m) profile and the very low rate constant for benzoyl-citrulline ethyl ester reveal a new kinetically influential group ionizing below the pK(a) of the active site histidine and indicate that the positive charge of arginine is essential for effective catalysis. (iv) The enzyme is inhibited by high concentrations of substrate. The mechanism of inhibition markedly varies with the reaction conditions. (v) The optimum temperature for the reactions of amide substrates is unusually low, slightly below 25 degrees C, whereas with benzoyl-arginine ethyl ester a linear Eyring plot is obtained up to 39 degrees C. The positive entropies of activation point to substantial reorganization of water molecules upon substrate binding.     

Recombinant expression, purification, and kinetic characterization of chondroitinase AC and chondroitinase B from Flavobacterium heparinum

Glycosaminoglycans (GAGs) are a family of complex polysaccharides involved in a diversity of biological processes, ranging from cell signaling to blood coagulation. Chondroitin sulfate (CS) and dermatan sulfate (DS) comprise a biologically important subset of GAGs. Two of the important lyases that degrade CS/DS, chondroitinase AC (EC 4.2.2.5) and chondroitinase B (no EC number), have been isolated and cloned from Flavobacterium heparinum. In this study, we outline an improved methodology for the recombinant expression and purification of these chondroitinases, thus enabling the functional characterization of the recombinant form of the enzymes for the first time. Utilizing an N-terminal 6x histidine tag, the recombinant chondroitinases were produced by two unique expression systems, each of which can be purified to homogeneity in a single chromatographic step. The products of exhaustive digestion of chondroitin-4SO(4) and chondroitin-6SO(4) with chondroitinase AC and dermatan sulfate with chondroitinase B were analyzed by strong-anion exchange chromatography and a novel reverse-polarity capillary electrophoretic technique. In addition, the Michaelis-Menten parameters were determined for these enzymes. With chondroitin-4SO(4) as the substrate, the recombinantly expressed chondroitinase AC has a K(m) of 0.8 microM and a k(cat) of 234 s(-1). This is the first report of kinetic parameters for chondroitinase AC with this substrate. With chondroitin-6SO(4) as the substrate, the enzyme has a K(m) of 0.6 microM and a k(cat) of 480 s(-1). Recombinantly expressed chondroitinase B has a K(m) of 4.6 microM and a k(cat) of 190 s(-1) for dermatan sulfate as its substrate. Efficient recombinant expression of the chondroitinases will facilitate the structure-function characterization of these enzymes and allow for the development of the chondroitinases as enzymatic tools for the fine characterization and sequencing of CS/DS.     

Kinetics and comparative reactivity of human class I and class IIb histone deacetylases

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of acetylated lysine residues on histone proteins. They operate in biological systems as part of multiprotein corepressor complexes. To understand the reactivity of isolated HDACs and the contribution of cofactor binding to reactivity, the reaction kinetics of isolated, recombinant human HDACs 1, 2, 3, 6, 8, and 10 were measured using a novel, continuous protease-coupled enzyme assay. Values of k(cat) and k(cat)/K(m) and the pH dependence of these values were determined for the reactions of each isozyme with acetyl-Gly-Ala-(N(epsilon)-acetyl-Lys)-AMC. Values of k(cat) spanned the range of 0.006-2.8 s(-1), and k(cat)/K(m) values ranged from 60 to 110000 M(-1) s(-1). The pH profiles for both k(cat) and k(cat)/K(m) were bell-shaped for all of the HDAC isozymes, with pH optima at approximately pH 8. Values of K(i) for the inhibitor trichostatin A were determined for each isozyme. The inhibition constants were generally similar for all HDAC isozymes, except that the value for HDAC8 was significantly higher than that for the other isozymes. The reaction of HDAC8 with an alternative substrate was performed to assess the steric requirements of the HDAC8 active site, and the effect of phosphorylation on HDAC1 activity was examined. The results are discussed in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acetyllysine hydrolysis by these enzymes.     

The conserved R166 residue of ALDH5A (succinic semialdehyde dehydrogenase) has multiple functional roles

ALDH5 (aka succinic semialdehyde dehydrogenase) is a NAD(+)-dependent aldehyde dehydrogenase crucial for the proper removal of the GABA metabolite succinic semialdehyde (SSA). All known ALDH5 family members contain the conserved amino acid sequence "MITRK". Our studies of rat ALDH5A indicate that residue R166 in this sequence may play a role in the substrate specificity of ALDH5A for the gamma-carboxylated succinic semialdehyde versus other aliphatic and aromatic aldehydes including acetaldehyde and benzaldehyde. We tested the hypothesis that the R166 residue regulates aldehyde specificity by utilizing rat ALDH5A wild-type (R166wt) and R166K, R166H, R166A, and R166E mutants. The V(MAX) using SSA fell whereas the K(M) for SSA increased for all mutants analyzed yielding k(cat)/K(M) (s(-1)/microM) ratios of 52.3 (R166wt), 5.5 (R166K), 0.01 (R166H), 0.008 (R166E), and 0.004 (R166A). Utilization of acetaldehyde by the R166H mutant was similar to R166wt with k(cat)/K(M)'s of 0.003 and 0.002, respectively. Almost no activity towards acetaldehyde was noted for the R166E and R166A mutants. Unexpectedly, the K(M) for NAD(+) changed: 21 microM (R166wt), 81 microM (R166K), 63 microM (R166H), 35 microM (R166E) and 44 microM (R166A). As release of NADH can be a rate-limiting step for ALDH activity, NADH binding was evaluated for R166wt and R166H enzymes. The K(D) of NADH for R166H (0.9 microM) was 11-fold less than that of ALDH5A wt (10.3 microM) and possibly explains the increase in the K(M) for NAD(+). Furthermore, data using R166K and R166H mutants demonstrate that inhibition of enzyme activity by low pH is regulated in part by the R166 residue. Our data indicate that the R166 residue of ALDH5A regulates multiple enzymatic functions.     

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.     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

A functional comparison of ovine and porcine trypsins

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.     

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.     

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10?3 M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s?1, and 1.2 +/- 0.105 × 10? M?1s?1, respectively. The L-asparaginase activity was reduced in the presence of Mn2?, Zn2?, Ca2?, and Mg2? metal ions for about 52% to 31%. In addition, we found that NH??, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.     

Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis determining tumor malignancy. The aim of this study was to elucidate peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to tumor. All carcinoma specimens expressed the E7 HPV-16 gene. Results obtained indicate that the increased expression of MMP-1 and MMP-9 and low expression of TIMP-1 and TIMP-2 make the main contribution to the destructive (invasive) potential of SCC. Changes in MMP-2 expression are less important. In the specimens of morphologically normal tissue adjacent to the tumor, substantial expression of MMP-1, MMP-2 and MMP-9 was registered. This expression appears to make additional contribution to the tumor destructive potential.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.