Multiple proteins related to the soluble galactose-binding animal lectin revealed by a monoclonal anti-lectin antibody.

An enzyme catalysing the O-methylation of isobutyraldoxime by S-adenosyl-L-methionine was isolated from Pseudomonas sp. N.C.I.B. 11652. The enzyme was purified 220-fold by DEAE-cellulose chromatography, (NH4)2SO4 fractionation, gel filtration on Sephadex G-100 and chromatography on calcium phosphate gel. Homogeneity of the enzyme preparation was confirmed by isoelectric focusing on polyacrylamide gel and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme showed a narrow pH optimum at 10.25, required thiol-protecting agents for activity and was rapidly denatured at temperatures above 35 degrees C. The Km values for isobutyraldoxime and S-adenosyl-L-methionine were respectively 0.24 mM and 0.15 mM. Studies on substrate specificity indicated that attack was mainly restricted to oximes of C4-C6 aldehydes, with preference being shown for those with branching in the 2- or 3-position. Ketoximes were not substrates for the enzyme. Gel filtration on Sephadex G-100 gave an Mr of 84 000 for the intact enzyme, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated an Mr of 37 500, suggesting the presence of two subunits in the intact enzyme. S-Adenosylhomocysteine was a powerful competitive inhibitor of S-adenosylmethionine, with a Ki of 0.027 mM. The enzyme was also susceptible to inhibition by thiol-blocking reagents and heavy-metal ions. Mg2+ was not required for maximum activity.     

Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.     

Immobilization of oxalate decarboxylase to Eupergit and properties of the immobilized enzyme

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5?mol/L (NH(4))(2)SO(4), room temperature, and shaking at 30?rpm for 24?hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44?U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.     

Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants

Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C(4) species), compared to the pattern in Pisum sativum (a C(3) plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C(4)) was 45 degrees C compared to 30 degrees C in P. sativum (C(3)). A. hypochondriacus (C(4)) lost nearly 70% of PEPC activity on exposure to a low temperature of 15 degrees C, compared to only about a 35% loss in the case of P. sativum (C(3)). Thus, the C(4) enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C(3) species. As the temperature was raised from 15 degrees C to 50 degrees C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C(4) plants (45%) was more than that in C(3) species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C(4) plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C(4)) and P. sativum (C(3)) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C(3) and C(4) plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C(4)-PEPC.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

Evolution of C(4) phosphoenolpyruvate carboxylase in Flaveria: determinants for high tolerance towards the inhibitor L-malate

During the evolution of angiosperms, C4 phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C3 isozymes. To identify the evolutionary alterations which are responsible for C4-specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr C4 and its ortholog from the closely related C3 plant Flaveria pringlei Gand. were examined using reciprocal enzyme chimeras. The main determinants for a high tolerance towards L-malate were located in the C-terminal region of the C4 enzyme. The effect of interchanging the region between amino acids 296 and 437 was strongly dependent upon the activation of the enzyme by glucose-6-phosphate. This confirms earlier observations that this region is important for the regulation of the enzyme by glucose-6-phosphate and that it harbours determinants for the different response of the C3 and the C4 enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C4-specific amino acid, a serine in the C-terminal part of the enzyme, is not involved in conferring an increased L-malate tolerance to the C4 enzyme.     

Importance of the prime subsites of the C1s protease of the classical complement pathway for recognition of substrates

The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)' substrate residues of C2 and C4 conferred greater affinity of substrate for enzyme and also induced a sigmoidal dependence of enzyme velocity on substrate concentration. This indicates that the substrate gave rise to homotropic positive cooperative behavior in the enzyme. When C1s was in complex with C1q and C1r, as would occur under physiological conditions, the same behavior was observed, indicating that this mechanism is relevant in the complement pathway in vivo. We further investigated the requirements of C1s for prime side amino acids by examining a substrate library in which each of the P(1)'-P(4)' positions had been substituted by different classes of amino acids. This revealed that the P(1)' position was a major determinant of the selectivity of the enzyme, while certain substitutions at the P(1)'-P(4)' positions abolished the allosteric behavior, indicating that contact residues at these positions in the C1s enzyme must mediate the cooperativity. The studies reported here highlight the importance of prime subsites in C1s for interaction with its cognate substrates in the complement pathway and therefore yield greater understanding of the mechanism of interaction between this vital protease and its physiological substrates.     

The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia -- implications for the evolution of C4 photosynthesis

C4 phospho enolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K(0.5)-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K(0.5)-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k(cat). We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.     

New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus.

1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.     

Mutagenesis of the active site lysine 221 of the pyruvate kinase from Bacillus stearothermophilus

Lysine 221 of the pyruvate kinase from Bacillus stearothermophilus was mutated to arginine, leucine, asparatic acid and cysteine. All the mutated enzymes were 10(4) to 10(5) times less active than the wild-type enzyme. The cysteine-free enzyme C9S/C268S, and the enzyme C9S/C268S/K221C, which possessed a unique sulfhydryl group at position 221, were prepared. The former had comparable activity to the wild-type enzyme and the latter was 10(4) times less active. These enzymes were denatured and renatured after aminoethylation. The C9S/C268S/K221C enzyme failed to regain its activity when renatured without aminoethylation; but when it was renatured after aminoethylation, it regained 4.5% of the activity of the C9S/C268S enzyme. This evidence suggests the importance of the Lys221 for the pyruvate kinase activity. The kinetic parameters of the S-aminoethylated C9S/C268S/K221C enzyme suggest that it has decreased affinity for phosphoenolpyruvate.     

Characterization of an unusual cold-active beta-glucosidase belonging to family 3 of the glycoside hydrolases from the psychrophilic isolate Paenibacillus sp. strain C7

We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside) only below 30 degrees C, an indication of cold-active beta-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for beta-galactosidase activity, belonged to glycoside hydrolase family 3, a family of beta-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-beta-D-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-beta-D-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35 degrees C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-beta-glucosides such as helicin, MUG (4-methylumbelliferyl-beta-D-glucopyranoside), esculin, indoxyl-beta-D-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 beta-glucosidases. The hydrolysis of a variety of aryl-beta-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-beta-D-glucoside may make it useful for producing indigo.     

Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. Neutrophils and lymphocytes hardly metabolized LTC4, whereas macrophages rapidly converted LTC4 to LTD4. The LTC4-metabolizing enzyme of macrophages was present in the membrane fraction but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (greater than 90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC4 to LTD4, on the cell surface membrane.     

Observation by 13C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by 13C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using [2-13C]PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP (152 ppm) and EPSP bound to the enzyme in a ternary complex with phosphate (161 ppm). These peak assignments account for our quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted; the intermediate could be isolated by quenching with either triethylamine or 0.2 N KOH, allowing direct quantitation of the species bound to the enzyme. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal [Leo et al. (1990) J. Am. Chem. Soc. (in press)]. The rate of formation of the EPSP ketal was very slow, 3.3 X 10(-5) s-1, establishing that it is a side product of the normal enzymatic reaction, probably arising as a breakdown product of the tetrahedral intermediate. A slow formation of pyruvate was also observed and is attributable to the enzymatic hydrolysis of EPSP, with 5% of the enzyme sites occupied by EPSP and hydrolyzing EPSP at a rate of 4.7 X 10(-4) s-1. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, an NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of C5-dicarboxylic substrate to aspartate aminotransferase: implications for the conformational change at the transaldimination step

The mechanism for the reaction of aspartate aminotransferase with the C4 substrate, l-aspartate, has been well established. The binding of the C4 substrate induces conformational change in the enzyme from the open to the closed form, and the entire reaction proceeds in the closed form of the enzyme. On the contrary, little is known about the reaction with the C5 substrate, l-glutamate. In this study, we analyzed the pH-dependent binding of 2-methyl-l-glutamate to the enzyme and showed that the interaction between the amino group of 2-methyl-l-glutamate and the pyridoxal 5'-phosphate aldimine is weak compared to that between 2-methyl-l-aspartate and the aldimine. The structures of the Michaelis complexes of the enzyme with l-aspartate and l-glutamate were modeled on the basis of the maleate and glutarate complex structures of the enzyme. The result showed that l-glutamate binds to the open form of the enzyme in an extended conformation, and its alpha-amino group points in the opposite direction of the aldimine, while that of l-aspartate is close to the aldimine. These models explain the observations for 2-methyl-l-glutamate and 2-methyl-l-aspartate. The crystal structures of the complexes of aspartate aminotransferase with phosphopyridoxyl derivatives of l-glutamate, d-glutamate, and 2-methyl-l-glutamate were solved as the models for the external aldimine and ketimine complexes of l-glutamate. All the structures were in the closed form, and the two carboxylate groups and the arginine residues binding them are superimposable on the external aldimine complex with 2-methyl-l-aspartate. Taking these facts altogether, it was strongly suggested that the binding of l-glutamate to aspartate aminotransferase to form the Michaelis complex does not induce a conformational change in the enzyme, and that the conformational change to the closed form occurs during the transaldimination step. The hydrophobic residues of the entrance of the active site, including Tyr70, are considered to be important for promoting the transaldimination process and hence the recognition of the C5 substrate.     

Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment

Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4 degrees C, pH 5 at 60 degrees C, pH 7 at 55 degrees C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose.     

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.