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PubMed1987

Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells.

We have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO4/PAGE of 60 and 57 kDa. The minor 60-kDa polypeptide is a glycosylated form of the major 57-kDa protein containing N-linked complex oligosaccharides. Zymogen activation by trypsin results in the removal of 84 amino acids from the amino terminus of the enzyme generating a 45-kDa active enzyme species. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively (1-2 micrograms per 10(6) cells per 24 hr). Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. Our data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

Amino Acid Sequence↗
PubMed1982

Enzymatic depolymerization of heparin-related polysaccharides. Substrate specificities of mouse mastocytoma and human platelet endo-beta-D-glucuronidases.

Two endo-beta-D-glucuronidases acting on heparin-related polysaccharides were investigated: a mouse mastocytoma enzyme previously implicated in the postbiosynthetic modification of the heparin proteoglycan and a human platelet-derived enzyme, capable of degrading heparan sulfate as well as heparin (with elimination of anticoagulant activity). The mastocytoma enzyme was found to depolymerize a heparin precursor polysaccharide containing both N- and O-sulfate groups to fragments somewhat smaller in molecular size than commercially available heparin. In contrast, another heparin precursor species, containing N- but no O-sulfate groups was resistant to degradation. Furthermore, incubation of the mastocytoma endoglucuronidase with a heparin octasaccharide having high affinity for antithrombin failed to cleave the beta-glucuronidic linkage in the antithrombin-binding region. Previous studies established that the platelet endoglucuronidase can degrade the exclusively N-sulfated as well as the N- and O-sulfated heparin precursor polysaccharides (Oldberg, A., Heldin, C.-H., Wasteson, A., Busch, C., and Höök, M. (1980) Biochemistry 19, 5755-5762). This enzyme has now been found to attack also the beta-glucuronidic linkage in the antithrombin-binding region of the heparin molecule. The loss of bio-affinity resulting from cleavage of this linkage in the antithrombin-binding heparin octasaccharide was utilized to construct a sensitive, specific, and simple assay method for the platelet endoglucuronidase. It is concluded that the platelet endoglucuronidase has a lower degree of substrate specificity than has the mastocytoma enzyme, in reflection of the surmised functional roles of the two enzymes.

Animals↗
PubMed1997

CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis.

Proteolysis mediated by the anaphase-promoting complex (APC) triggers chromosome segregation and exit from mitosis, yet its regulation is poorly understood. The conserved Cdc20 and Cdh1 proteins were identified as limiting, substrate-specific activators of APC-dependent proteolysis. CDC20 was required for the degradation of the APC substrate Pds1 but not for that of other APC substrates, such as Clb2 and Ase1. Conversely, cdh1Delta mutants were impaired in the degradation of Ase1 and Clb2 but not in that of Pds1. Overexpression of either CDC20 or CDH1 was sufficient to induce APC-dependent proteolysis of the appropriate target in stages of the cell cycle in which substrates are normally stable.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1983

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1.

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.

Animals↗
PubMed1983

Substrate specificity and other properties of the beta-D-galactosidase from Aspergillus niger.

beta-D-Galactosidase from Aspergillus niger was purified by conventional techniques, including the repeated use of chromatography on hydroxylapatite. The final preparation represented a 112-fold purification, with a 22% yield. The specific activity of the purified enzyme was 72 mumol of D-galactose released/min/mg of protein, using p-nitrophenyl beta-D-galactopyranoside as the substrate. The substrate specificity of the enzyme was studied by using saccharides having structural linkages similar to those found in naturally occurring glycoconjugates. At substrate concentrations of 5mM, the beta-D-galactosidase efficiently hydrolyzed beta-Gal-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 3)-Gal, beta-Gal-(1 leads to 3)-beta-Gal-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 3)-alpha-Gal-1 leads to OC6H4NO2-p, at rates of 63, 53, 65, and 29 mumol/min/mg of protein, respectively. Slower hydrolysis was observed for beta-Gal-(1 leads to 4)-beta-Glc, beta-Gal-(1 leads to 4)-beta-GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, with rates of 10, 13 and 9 mumol/min/mg of protein, respectively. Poorly hydrolyzed, at rates 1/300th of that of beta-Gal-1 leads to OC6H4NO2-p, were synthetic substrates having D-galactose attached beta-(1 leads to 3)- to either GalNAc or GlcNAc. The Km value for beta-D-galactosidase with beta-Gal-(1 leads to 4)-beta-GlNAc-1 leads to OC6H4NO2-p was approximately 20 times that with beta-Gal-1 leads to OC6H4NO2-p. The beta-D-galactosidase of A. niger has a molecular weight of 300,000, as demonstrated by gel-filtration chromatography. Sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis indicated a single subunit having a molecular weight of 130,000.

Aspergillus niger↗
PubMed1994

D-amino acid residues as substrate specificity determinants for casein kinase II.

A set of diastereomeric peptides RRRDDDSDDD each with a corresponding D-amino acid residue successively in every position (except the arginines) was tested as substrates for casein kinase II. It was found that the D-serine containing peptide was not detectably phosphorylated. The replacements at the positions +1, -1 and +2 decreased the kII values more than 60, 30 and 20 times, respectively. The effect of the L/D replacements decreased with increased distance from the serine. The D-amino acid scan used herein seems to be a helpful complementary tool for studies of substrate specificity determinants for different protein kinases.

Amino Acid Sequence↗
PubMed1989

Substrate specificity of bleomycin hydrolase.

Bleomycin (BLM) hydrolase is believed to protect both malignant and normal tissue from the toxicity of the antitumor drug BLM. Little is known about the substrate specificity of BLM hydrolase. Thus, we developed ion-paired reverse phase high speed liquid chromatography systems to assay for the metabolism of several BLM analogs. We found that BLM A2, BLM B2, tallysomycin S10b (TLM S10b), peplomycin (PEP), butylamino-3-propylamino-3-propylamine bleomycin (BAPP), deglyco bleomycin A2 (dgBLM A2) and bleomycinic acid were each metabolized by rabbit lung BLM hydrolase to a single metabolite. When compared to their corresponding parent compounds, these metabolites were 6- to 35-fold less potent in their ability to inhibit the proliferation of A-253 human head and neck squamous carcinoma cells in culture. Furthermore, we found that substitutions in various regions of the BLM molecule greatly affected the kinetic parameters of BLM hydrolase. For example, the Km with BLM B2 (0.056 +/- 0.005 mM) was 15-fold lower than that seen with BLM A2 (0.83 +/- 0.11 mM). In contrast, the Vmax was not affected markedly by these terminal amine substitutions but was influenced greatly by deletion of the carbohydrate groups of BLM. For example, a 4-fold higher Vmax was observed with dgBLM A2 compared to BLM A2. Thus, these results demonstrate that BLM hydrolase can recognize and metabolize a broad spectrum of BLM analogs regardless of their structural features. This enzymatic conversion resulted in the inactivation of the BLMs as demonstrated by a substantial decrease in their cytotoxicity. Furthermore, the terminal amine and carbohydrate regions, respectively, dictate the apparent affinity and the rate of metabolism of BLM hydrolase substrates.

Animals↗
PubMed1999

Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.

DNA Adducts↗
PubMed1998

X-ray analysis of the NMC-A beta-lactamase at 1.64-A resolution, a class A carbapenemase with broad substrate specificity.

The treatment of infectious diseases by penicillin and cephalosporin antibiotics is continuously challenged by the emergence and the dissemination of the numerous TEM and SHV mutant beta-lactamases with extended substrate profiles. These class A beta-lactamases nevertheless remain inefficient against carbapenems, the most effective antibiotics against clinically relevant pathogens. A new member of this enzyme class, NMC-A, was recently reported to hydrolyze at high rates, and hence destroy, all known beta-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-A resolution, and reveals modifications in the topology of the substrate-binding site. While preserving the geometry of the essential catalytic residues, the active site of the enzyme presents a disulfide bridge between residues 69 and 238, and certain other structural differences compared with the other beta-lactamases. These unusual features in class A beta-lactamases involve amino acids that participate in enzyme-substrate interactions, which suggested that these structural factors should be related to the very broad substrate specificity of this enzyme. The comparison of the NMC-A structure with those of other class A enzymes and enzyme-ligand complexes, indicated that the position of Asn-132 in NMC-A provides critical additional space in the region of the protein where the poorer substrates for class A beta-lactamases, such as cephamycins and carbapenems, need to be accommodated.

Amino Acid Sequence↗
PubMed1987

[Purification and substrate specificity of BcmI restriction endonuclease].

A strain producing the new specific restriction endonuclease BcmI has been found in the Bacillus generum. The enzyme has been purified by chromatography on the blue sepharose, phosphocellulose PII, heparin sepharose. The analogous purification has been obtained when the blue sepharose has been substituted for the orange sepharose, the home produced sorbent. The BcmI enzyme has been shown by the substrate specificity definition to be an isoschizomer of the restriction endonuclease ClaI.

Bacillus↗
PubMed1987

Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver.

Several GM3 derivatives have been synthesized. Among them were lyso-GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.

Animals↗
PubMed1998

Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid CoA thioester.

The substrate specificity of rat liver peroxisomal 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-18O3]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on a steroid nucleus of 3,7,12-trioxo-5 beta-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and 18O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed delta 24-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The delta 24-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.

Animals↗
PubMed1994

Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite.

Interest in the active site specificity of human cathepsin D stems from the search for specific therapeutic agents against many of the sequentially and structurally homologous members of the aspartic proteinase family. The work presented here examined one amino acid in the cathepsin D sequence, located in the S2 subsite, which contributes substantially to the specificity of enzyme-ligand interactions at the enzyme active site. Previous studies reported on the specificity of binding and catalysis by native and recombinant human cathepsin D explored through kinetic studies using a systematic series of synthetic substrates. Utilizing a rule-based molecular model of human cathepsin D, Met287 was suggested as a candidate for mutagenesis to further explore selectivity within the S2 subsite of the cathepsin D active site. Met287 mutant derivatives of human cathepsin D were designed, expressed and characterized in kinetic studies. Native cathepsin D accommodates large hydrophobic residues in the P2 position of a substrate; positively charged residues in P2 are not favorable for catalysis. It was demonstrated that altering Met287 of human cathepsin D to more polar amino acids produced active mutant enzymes with significantly altered substrate specificity.

Amino Acid Sequence↗
PubMed1995

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue.

Two residues, K89 and S380, thought to interact with the gamma-carboxyl group of the substrate L-glutamate, have been altered by site-directed mutagenesis of clostridial glutamate dehydrogenase (GDH). The single mutants K89L and S380V and the combined double mutant K89L/S380V were constructed. All three mutants were satisfactorily overproduced in soluble form. However, only the K89L mutant was retained by the dye column normally used in purifying the wild-type enzyme. All three mutant enzymes were purified to homogeneity and tested for substrate specificity with 24 amino acids. The single mutant S380V showed no detectable activity. The alternative single mutant K89L showed an activity towards L-glutamate that was decreased nearly 2000-fold compared with wild-type enzyme, whereas the activities towards the monocarboxylic substrates alpha-aminobutyrate and norvaline were increased 2- to 3-fold. A similar level of activity was obtained with methionine (0.005 U/mg) and norleucine (0.012 U/mg), neither of which give any activity with the wild-type enzyme under the same conditions. The double mutant showed decreased activity with all substrates compared with the wild-type GDH. In view of its novel activities, the K89L mutant was investigated in greater detail. A strictly linear relationship between reaction velocity and substrate concentration was observed up to 80 mM L-methionine and 200 mM L-norleucine, implying very high Km values. Values of kcat/Km for L-methionine and L-norleucine were 6.7 x 10(-2) and 0.15 s-1 M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Characterization of chimeric pterin-dependent hydroxylases: contributions of the regulatory domains of tyrosine and phenylalanine hydroxylase to substrate specificity.

Tyrosine and phenylalanine hydroxylases contain homologous catalytic domains and dissimilar regulatory domains. To determine the effects of the regulatory domains upon the substrate specificities, truncated and chimeric mutants of tyrosine and phenylalanine hydroxylase were constructed: Delta117PAH, the C-terminal 336 amino acid residues of phenylalanine hydroxylase; Delta155TYH, the C-terminal 343 amino acid residues of tyrosine hydroxylase; and 2 chimeric proteins, 1 containing the C-terminal 331 residues of phenylalanine hydroxylase and the N-terminal 168 residues of tyrosine hydroxylase, and a second containing the C-terminal 330 residues of tyrosine hydroxylase and the 122 N-terminal residues of phenylalanine hydroxylase. Steady-state kinetic parameters with tyrosine and phenylalanine as substrate and the need for pretreatment with phenylalanine for full activity were determined. The truncated proteins showed low binding specificity for either amino acid. Attachment of either regulatory domain greatly increased the specificity, but the specificity was determined by the catalytic domain in the chimeric proteins. All three proteins containing the catalytic domain of phenylalanine hydroxylase were unable to hydroxylate tyrosine. Only wild-type phenylalanine hydroxylase required pretreatment with phenylalanine for full activity with tetrahydrobiopterin as substrate.

Amino Acid Sequence↗
PubMed1988

Myristoyl CoA:protein N-myristoyltransferase activities from rat liver and yeast possess overlapping yet distinct peptide substrate specificities.

A variety of eukaryotic viral and cellular proteins possesses an NH2-terminal N-myristoylglycine residue important for their biological functions. Recent studies of the primary structural requirements for peptide substrates of the enzyme responsible for this modification in yeast demonstrated that residues 1, 2, and 5 play a critical role in enzyme: ligand interactions (Towler, D. A., Adams, S. P., Eubanks, S. R., Towery, D. S., Jackson-Machelski, E., Glaser, L., and Gordon J. I. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 2708-2812). This was determined by examining as substrates a series of synthetic peptides whose sequences were systematically altered from a "parental" peptide derived from the known N-myristoylprotein bovine heart cyclic AMP-dependent protein kinase (A kinase) catalytic subunit. We have now extended these studies in order to examine structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT). The interaction between yeast NMT and the side chain of residue 5 in peptide ligands is apparently sterically constrained, since Thr5 is unable to promote the very high affinity binding observed with a Ser5 substitution. A substrate hexapeptide core has been defined which contains much of the information necessary for recognition by this lower eukaryotic NMT. Addition of COOH-terminal basic residues to this hexapeptide enhances peptide binding, while COOH-terminal acidic residues destabilize NMT: ligand interactions. Based on the results obtained from our in vitro studies of over 80 synthetic peptides and yeast NMT, we have identified a number of potential N-myristoylproteins from searches of available protein databases. These include hepatitis B virus pre-S1, human SYN-kinase, rodent Gi alpha, and bovine transducin-alpha. Peptides corresponding to the NH2-terminal sequences of these proteins and several known N-myristoylproteins were assayed using yeast NMT as well as partially purified rat liver NMT. While a number of the synthetic peptides exhibited similar catalytic properties with the yeast and mammalian enzymes, surprisingly, the SYN-kinase, Gi alpha, and transducin-alpha peptides were N-myristoylated by rat NMT but not by yeast NMT. This suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.

Acyl Coenzyme A↗
PubMed2004

Family shuffling of expandase genes to enhance substrate specificity for penicillin G.

Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

Actinobacteria↗
PubMed2001

Importance of amino acid residue 474 for substrate specificity of canine and human cytochrome p450 3A enzymes.

Canine cytochromes P450 3A12 and 3A26 are identical in sequence at 481 of 503 amino-acid positions but exhibit different substrate specificities. A recent study utilizing chimeric enzymes and site-directed mutagenesis identified three residues (187, 368, and 369) that contribute to differences in steroid hydroxylation and also indicated the presence of additional determinants of specificity among the 44 carboxyl terminal residues. Therefore, three 3A26 multiple mutants (I187T-S368P-V369I-S467P, I187T-S368P-V369I-S474P, and I187T-S368P-V369I-R476K-I477L-T479A-R480Q) were constructed. Insertion of 3A12 residue Pro-474 into 3A26 I187T-S368P-V369I resulted in metabolite profiles with testosterone, androstenedione, and progesterone very similar to 3A12. Substitution of Pro-474 with Ser in P450 3A12 or human 3A4 significantly increased 2beta-hydroxylase activity with all three steroids. Residue 474 was also found to be an important contributor to diazepam metabolism by the canine and human enzymes. The results provide further evidence for the role of steric constraints exerted by the enzyme in P450 3A-mediated oxidations.

Amino Acid Substitution↗