Variation In Substrate Specificity Research Articles

Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km=3740+/-413 M-1.s-1 versus 472+/-72.4 M-1.s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki=208200+/-36000 M-1. s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l- prolin e, kinact/Ki=199200+/-32900 M-1.s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer (review).

Lysosomal cysteine proteinases, the cathepsins (Cats) belong to the papain family of proteinases, sharing a similar protein structure and mechanism of action. Subtle structural differences between these enzymes give rise to important variations in substrate specificity and specificity of inhibition by their endogenous inhibitors, the cystatins, stefins and kininogens under physiological and pathological conditions. Alterations in their expression, processing and localization have been observed at various levels in malignant human tumor tissue compared to normal and benign tissue counterparts. We have proposed that an imbalance between cathepsins and cystatins, associated with metastatic tumor cell phenotype, may facilitate tumor cell invasion and metastasis. The results of clinical investigations on cysteine cathepsins and their endogenous inhibitors in human breast, lung, brain and head and neck tumors, as well as in body fluids of ovarian, uterine, melanoma and colorectal carcinoma bearing patients, have shown that these molecules are highly predictive for the length of survival and may be used for assessment of risk of relapse and death for cancer patients. Their application for diagnosis, follow-up and the anticancer therapy has also been proposed.

Molecular modelling of human CYP2C subfamily enzymes CYP2C9 and CYP2C19: rationalization of substrate specificity and site-directed mutagenesis experiments in the CYP2C subfamily

1. The results of molecular modelling of human CYP2C isozymes, CYP2C9 and CYP2C19, are reported based on an alignment with a bacterial form of the enzyme, CYP102. 2. The three-dimensionalstructures of the CYP2C enzymes are consistent with known experimental evidence from site-directed mutagenesis, antibody recognition and regiospecificity of substrate metabolism. 3. The variations in substrate specificity between CYP2C9 and CYP2C19 can be rationalized in terms of single amino acid residue changes within the putative active site region, of which I99H appears to be the most significant.

Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.

Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.

The catalytic subunit of cAMP-dependent protein kinase from Ascaris suum. The cloning and structure of a novel subtype of protein kinase A.

A complete cDNA clone encoding the catalytic subunit of cAMP-dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 amino acids is 48% identical with the sequence of mouse C alpha subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N-terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N-terminal region and an acidic amino acid near the C-terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N-terminal and C-terminal parts of the protein. There is strong evidence that a single gene encodes cAMP-dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X-ray structure of the mammalian enzyme suggest a high degree of conservation in the three-dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952-957] and the mammalian enzyme.

The structure of the transglutaminase 1 enzyme. Deletion cloning reveals domains that regulate its specific activity and substrate specificity.

Transglutaminase 1 (TGase1) is one of three known enzymes involved in terminal differentiation in stratified squamous epithelia, possibly in the formation of a cornified cell envelope. Because the intact enzyme is particularly difficult to isolate in quantity from keratinocytes for characterization, comparatively little is known about its properties. We have expressed the full-length as well as a series of deletion forms of this enzyme in a bacterial system and analyzed their enzymatic properties. The specific activity of the full-length enzyme isolated and purified from the bacterial lysate was comparable to that of the native enzyme of keratinocytes. Analysis of several deletion constructs demonstrated that removal of the first 60-109 residues, which include sequences involved in membrane association, results in upwards of a 10-fold increase in the specific activity. Deletions beyond residue 109, into sequences conserved within the TGase family of proteins, result in loss of activity. Similarly, as many as 240 residues can be removed from its carboxyl-terminal end before activity is lost. Thus, a molecule of 466 residues, containing virtually only the conserved core sequences of TGases, retains a specific activity comparable to the intact enzyme. In addition, the various deletion forms display wide variations in substrate specificity toward a series of synthetic peptide substrates, designed from possible target TGase1 substrate proteins of epithelia. The data show that sequences between residues 62 and 92 are important in defining the substrate specificity of the TGase1 enzyme system. Furthermore, it may now be possible to design an enzyme with a defined substrate specificity. Together, these data suggest TGase1 has recruited additional sequences on its amino terminus in relation to other members of the TGase family, which have the net effect of permitting sequestration onto membranes, changing its specific activity and modifying its likely substrate specificities.

Proteolytic processing of the pro beta chain of beta-hexosaminidase occurs at basic residues contained within an exposed disulfide loop structure.

Lysosomal beta-hexosaminidase (EC 3.2.1.52) occurs as two major isozymes, Hex A (alpha beta) and Hex B (beta beta). The alpha and beta subunits are encoded by the HEXA and HEXB genes, respectively. Extensive homology in both the gene structures and deduced primary sequences demonstrate their common evolutionary origin. While undergoing similar proteolytic modifications in the lysosome, the pro beta polypeptide is additionally cleaved internally to produce the mature 24-30 kilodalton beta b and beta a chains. Previous data have suggested that this processing event occurs somewhere between residues Ser311 and Lys315. In this report we demonstrate that this area is located in a hydrophilic disulfide-loop structure (between Cys309 and Cys360). The cleavage event is prevented by the deletion through in vitro mutagenesis of the Arg312-Gln-Asn-Lys tetrapeptide or by its substitution with the aligned alpha residues (Gly-Ser-Glu-Pro). Reintroduction of either Arg312 or Lys315 reinstates the processing. Furthermore, we show that this area is not involved in lysosomal targeting of pro-Hex B, or in the increased stability or the variation in substrate specificity of the beta as compared with the alpha subunit. Our data suggest the presence of a novel lysosomal endoprotease. Like other endoproteases it is specific for basic amino acids; however, it cleaves on the amino-terminal side rather than the conventional carboxy-terminal side of such residues and then only if they are fully exposed to the lysosomal environment.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of bile acid CoA:Amino acid: N-acyltransferase (BAT) from four mammalian species

1. 1. Bile acid CoA:amino acid: N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity ( K m) and turnover number ( V max) of BAT activity. 4. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. 5. Immunoabsorption chromatography using an anti-human BAT antibody—Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.

Identification of thymidine nucleotidase and deoxyribonucleotidase activities among normal isozymes of 5'-nucleotidase in human erythrocytes.

The persistence of normal thymidine nucleotidase (ThyNase) activity in subjects with pyrimidine nucleotidase (PyrNase) deficiency suggested the possible existence of separate isozymes in normal human erythrocytes. This hypothesis was confirmed by studies of PyrNase-deficient individuals from five unrelated families. Erythrocytes deficient in PyrNase retained normal activity of an enzyme system preferentially active at pH 6.2 with a variety of 2'-deoxyribonucleoside 5'-monophosphate substrates, including those of uridine, thymidine, and cytidine. Lesser activities were observed with the corresponding ribonucleotides. Normal control hemolysates were also found capable of effectively dephosphorylating purine nucleotides (dAMP greater than AMP) when pH was lowered sufficiently from the pH 7.4-8.0 region commonly used in conventional assays. Variations in substrate specificity, pH optima, kinetics, and sensitivity to inactivation by Pb2+ indicated the existence of multiple 5'-nucleotidase isozymes in normal erythrocytes: PyrNase and deoxyribonucleotidase(s) that might function physiologically in the conversion of DNA-derived nucleotides to diffusible nucleosides. Evolution of such a unique 5'-nucleotidase suggests that normal erythroblast maturation and nuclear extrusion is accompanied by a degree of karyolysis sufficient to require dephosphorylation and clearance of DNA degradation products.

Host specificity in sponge-encrusting zoanthidea (Anthozoa: Zoantharia) of Barbados, West Indies

Six species of common Caribbean Zoanthidea, Parazoanthus swiftii, P. parasiticus, P. catenularis, P. puertoricense, Epizoanthus cutressi, and Epizoanthus sp., are virtually restricted to living on surfaces of reef-dwelling sponges. Quantitative surveys on Barbados reefs indicate that substrate specificity is relatively high among these zoanthids with three restricted to a single primary host sponge species and three restricted to three closely related sponges. One species, P. swiftii, exhibits a broad range of acceptable secondary substrates, due to its unique ability to execute migrational spread in the adult polyp stage. Variations in substrate specificity have been noted between island populations within the extensive Caribbean range and appear to be due to different species compositions of local sponge communities and slight differences in zoanthid larval settling specificities.

Carboxylesterases (EC 3.1.1). The source of variations in substrate specificity and properties of pig liver carboxylesterase

During the final stage of purification of pig liver carboxylesterase on CM-Sephadex, several enzyme activities are present in addition to the pig liver carboxylesterase reported from this laboratory (Horgan et al., 1969a). Variation in substrate specificity and specific activity of the material isolated from CM-Sephadex has been analysed with four substrates - methyl, ethyl and phenyl butyrates and butyrycholine. The sensitivity of various fractions to paraoxon, eserine and phenylmethanesulfonyl fluoride is also examined. The results support the conclusion that the variability of pig liver carboxylesterase reported from other laboratories is due to the heterogeneity here defined.

Studies on myrosinases III. Enzymatic properties of myrosinases from Sinapis alba and Brassica napus seeds

A comparative study was performed on some enzymatic properties of myrosinase isoenzymes (thioglucoside glucohydrolases, EC 3.2.3.1) from white mustard seed ( Sinapis alba) and rapeseed Brassica napus). The investigation comprised the determination of pH optima, temperature maxima and the enzymatic stabilities of the isoenzymes, when temperature, pH, buffer and time were varied. The effect of ascorbic acid on the kinetic constants K m and V was found to be different for the isoenzymes. Comparative studies were also made of the activities of the isoenzymes on various glucosinolates, and the myrosinase activities in a number of cultivars of B. napus, Brassica campestris and S. alba. The results indicate that there are both physicochemical and enzymatic differences between myrosinase isoenzymes and that a large amounty of variance exists. They are, however, likely to have the same genetic origin. No significant variation in substrate specificity was found among the isoenzymes.

Red cell acid phosphatase, phenolphthaleindiphosphate, and β‐naphtyl phosphate: variation in substrate specificity

Contrary to carlicr results it is found that β‐naphtyl phosphate may be used as a substrate for red cell acid phosphatase and that thereby isozymes are revealed that do not appear with phenolphtha‐lein diphosphate as substrate. Qualitative differences between the zymograms with the two substrates are interpreted as being due to variation in substrate specificity between isozymes. Quantitatively, the enzymatic activity is of the same order as regards these two substrates. Previous unsuccessful attempts to obtain a zymogram with naphtyl phosphate are shown to be due to inhibition of the enzyme by diazonium salts.

Red cell acid phosphatase, phenolphthalein diphosphate, and ß-naphtyl phosphate: variation in substrate specificity

Red cell acid phosphatase, phenolphthalein diphosphate, and ß-naphtyl phosphate: variation in substrate specificity