Comparison of in Vitro and in Vivo Activities Associated with the G6pd Allozyme Polymorphism in Drosophila Melanogaster

Earlier studies of the A and B allozymes at the G6pd locus show a differential ability of the genotypes to suppress the loss of viability associated with a low activity 6-phosphogluconate dehydrogenase mutation, 6Pgdlo1. This observation indicates a relatively lower activity for the A allozyme genotype, but it is not known if this level of suppression required a large difference in in vivo activity. To clarify this difference an analysis of the biochemical properties of the purified allozymes was carried out, as well as an analysis of the activity level associated with an original low activity P element-derived allele which had partially reverted and lost its suppression ability. G6PD activity and protein level were studied in 47 X chromosome lines from North America. The A genotype averages a 9% lower Vmax. From analysis of the correlation between G6PD activity and protein level it remains unclear whether the allozyme Vmax difference results from dissimilarity in protein level or kcat. At 25 degrees and physiological pH, comparative studies of the steady-state kinetics show the two purified allozyme variants differ significantly in their KM values for glucose-6-phosphate and NADP, and the K1 for NADPH. In aggregate these parameters predict the A genotype possesses a 20% lower in vitro catalytic efficiency. A partial revertant of a P element-derived low activity B variant, was shown to lose the ability to suppress 6Pgdlo1 low viability after acquiring only 60% of normal B activity. This last comparison shows the A genotype activity must be reduced in vivo by at least 40%.     

Mechanism-controlled stereospecificity. Acylation of subtilisin with enantiomeric alkyl and nitrophenyl ester substrates.

The activation parameters of acylation of subtilisin with alkyl and p-nitrophenyl esters of N-acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 10(4)--10(5) with methyl esters whereas it is only of the order of 10 with nitrophenyl esters. The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme. The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy-entropy compensation.     

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.     

Development of a newBacillus carboxyl esterase for use in the resolution of chiral drugs

We have screened a new enzyme for the resolution ofR, S-naproxen enantiomers. The enzyme is free of lipase activity, and possesses a very high stereo-specificity on S-naproxen [2-(6-methoxy-2-naphthyl)-propionic acid] esters and esters of related drugs. The primary structure of the enzyme, determined from the nucleotide sequence, shows limited homology with the catalytic site of lipases. The gene coding for the stereo-selective carboxylesterase has been cloned and expressed inBacillus subtilis. Using a multicopy vector and an additional strong promoter an efficient production process was developed.The enzyme was shown to be sensitive to very high concentrations of the products formed during the reaction it catalyses. To increase the resistance of the enzyme, lysine residues thought to be responsible for this phenomenon were replaced through site-directed mutagenesis. Enzymes with improved stability were obtained. An explanation is given in terms of a model in which a reaction of the acid moiety of naproxen with free lysine NH₂ groups is a major cause of inactivation.     

The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.

Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.     

Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K _m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k _cat) and the k _cat/K _m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s^?1/10,412–10,795 s^?1 M^?1 for AAT-2 zone and 4.6–7.0 s^?1/527–700 s^?1 M^?1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.     

Design,synthesis and biological activity of novel non-amidine factor Xa inhibitors. Part 1: P(1) structure-activity relationships of the substituted 1-(2-Naphthyl)-1H-pyrazole-5-carboxylamides

Based on DuPont Pharmaceuticals' monobenzamidine lead structure SN429, we have designed the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as a novel series of non-basic factor Xa inhibitors. We have discovered that the displacement of the benzamidine moiety with substituted 2-naphthyl structures not only results in highly potent factor Xa inhibitors, but also significantly increases their enzyme specificity and oral bioavailability.     

N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Rü61a, an alpha/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant

N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Rü61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.     

Isolation and kinetic analysis of the multiple forms of glyoxalase-I from human erythrocytes.

A simple electrophoresis procedure was developed to detect glyoxalase-I variants in human erythrocytes by using a direct stain for glyoxalase-I activity. The multiple forms of glyoxalase-I were purified from human erythrocytes by a procedure involving ethanol;chloroform treatment to remove hemoglobin and chromatography on blue dextran affinity columns. Glyoxalase-I from individuals with homozygous phenotypes (GLO-1 or GLO-2) was more labile in this purification procedure than was glyoxalase-I from individuals with the heterozygous phenotype (GLO 2-1). Nevertheless, the various glyoxalase-I allozymes are indistinguishable, kinetically. In addition, the glyoxalase-I allozymes from human erythrocytes are kinetically similar to glyoxalase-I from other mammalian sources in that they exhibit broad substrate specificity for the hemimercaptals of glutathione and aliphatic or aromatic α-ketoaldehydes and function by rate-determining cleavage of the hemimercaptal C-H bond, as reflected in a primary isotope effect.     

Structure-function studies of two novel UDP-GlcNAc C6 dehydratases/C4 reductases. Variation from the SYK dogma

Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu(589) residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp(70), Asp(149)-Lys(150), Cys(103)) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.     

Serine esterases: structural conservation during animal evolution and variability in enzymic properties in the genus Drosophila

Both general esterases and acetylcholinesterases have been shown to be members of a homologous superfamily of serine esterases. A comparison of N-terminal sequences demonstrates that esterase-4 and-5 from Drosophila mojavensis belong to this family as well, with esterase-6 and esterase-P from D. melanogaster being the closest relatives. In order to investigate the presence of immunologically related esterases in other Drosophila species, crude larval extracts from five species were applied to two immunoaffinity columns with antibodies directed against esterase-4 and esterase-5 from D. mojavensis. The substrate preference for either 1- or 2-naphthyl acetate was determined. Both esterase-4 and esterase-5 from D. mojavensis are normally specific for 2-naphthyl esters, but at least three of the cross-reacting esterases from the other species have a preference for 1-naphthyl esters. This difference in substrate preference is another example of the variability observed with Drosophila esterases.     

Esterase-6 and the pheromonal effects of cis-vaccenyl acetate in Drosophila melanogaster

In Drosophila melanogaster the polymorphic enzyme Est-6 and a male specific lipid, cis-vaccenyl acetate (cVA), are components of the seminal fluid. It has been suggested that cVA could be a physiological substrate for Est-6, constituting, together with the hypothetical hydrolytic product, cis-vaccenyl alcohol (cVOH), a pheromonal system active in the regulation of the sexual attractiveness of mated females. However, some doubt exists concerning the physiological conversion of cVA to cVOH in females. Est-6 is also known to affect sperm motility, sperm use and female fecundity. The results of a study on qualitative and quantitative effects of topical treatment of females with cis-vaccenyl acetate and cis-vaccenyl alcohol on courtship behaviour are presented. The inhibition of courtship produced by cVOH was more persistent; this was possibly related to its lower volatility. The hypothesis was tested that different Est-6 allozymes could produce different amounts of cis-vaccenyl alcohol in vivo by differential hydrolysis of cis-vaccenyl acetate, and thus affect the timing of remating. For this purpose highly homogeneous Est-6^S and Est-6^F homozygous lines, with almost certainly identical levels of Est-6 and cVA, were used. They were obtained after 100 generations of inbreeding and selection of the heterozygotes at each generation. The Est-6^S and Est-6^F products of the Est-6 structural gene did not differentially affect the timing of remating. This could imply that the two allozymes have similar catalytic properties. However, such results could be obtained even if cVA is not converted to cVOH in vivo by Est-6. The effects of Est-6 allozymes on sperm use after remating were also investigated. The general effect of second males was a very high elimination of first male fertilizations. No evidence for a different contribution of the two allozymes to the fitness through sperm predominance was obtained. Remating and sperm predominance experiments were performed at three temperatures: 18, 25 and 30° C. A more general—not species specific—role of cis-vaccenyl esters is suggested by preliminary results on their presence in the ejaculate of other Drosophila species.     

Sequence and phylogenetic analysis of viper venom serine proteases

Snakebites are a major neglected tropical disease responsible for as many as 95000 deaths every year worldwide. Viper venom serine proteases disrupt haemostasis of prey and victims by affecting various stages of the blood coagulation system. A better understanding of their sequence, structure, function and phylogenetic relationships will improve the knowledge on the pathological conditions and aid in the development of novel therapeutics for treating snakebites. A large dataset for all available viper venom serine proteases was developed and analysed to study various features of these enzymes. Despite the large number of venom serine protease sequences available, only a small proportion of these have been functionally characterised. Although, they share some of the common features such as a C-terminal extension, GWG motif and disulphide linkages, they vary widely between each other in features such as isoelectric points, potential N-glycosylation sites and functional characteristics. Some of the serine proteases contain substitutions for one or more of the critical residues in catalytic triad or primary specificity pockets. Phylogenetic analysis clustered all the sequences in three major groups. The sequences with substitutions in catalytic triad or specificity pocket clustered together in separate groups. Our study provides the most complete information on viper venom serine proteases to date and improves the current knowledge on the sequence, structure, function and phylogenetic relationships of these enzymes. This collective analysis of venom serine proteases will help in understanding the complexity of envenomation and potential therapeutic avenues.     

Digestion in vitro of erythritol esters by rat pancreatic juice enzymes

The mechanism of the digestion of erythritol esters was determined using rat pancreatic juice and purified pancreatic lipase (EC 3.1.1.3). Conditions of hydrolysis were used that would selectively activate or inactivate nonspecific lipase or lipase. It was shown that erythritol tetraoleate was hydrolyzed by nonspecific lipase but not by lipase. The initial digestion product was a triester, predominantly erythritol-1,2,3-trioleate. Thus, nonspecific lipase preferentially hydrolyzed the ester of a primary alcohol. In contrast to the results obtained with the tetraester, lipase could remove a fatty acid from the triester but the resulting erythritol-2,3-dioleate was not hydrolyzed by lipase. The selectivity of this hydrolysis and the inability to hydrolyze the diester are attributed to the known specificity of this enzyme to act only on esters of primary alcohols. Nonspecific lipase completely hydrolyzed erythritol tetraoleate to free erythritol in a stepwise manner. The relative rates of these reactions were tetraester --> triester --> diester --> monoester --> erythritol Because of the specificity of pancreatic lipase and the lack of specificity of nonspecific lipase it is likely that this latter enzyme is the primary agent for the hydrolysis of erythritol esters in the intact animal.     

The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase

Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions. Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit. Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa. If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential.     

Small-scale patterns of genetic variation in the mayfly Bungona narilla (Ephemeroptera: Baetidae) in rainforest streams, south-east Queensland

SUMMARY 1. Genetic structure of the mayfly Bungona narilla was examined using allozymes and a section of the cytochrome oxidase I gene.
2. The study had two major aims. The first was to determine whether patterns of genetic variation in mitochondrial DNA were similar to those found previously for allozymes, i.e. that more variation was evident among pools within a single stream than among streams. The mitochondrial DNA results were similar to those reported previously for allozymes, supporting the idea that larvae within any particular pool were unrepresentative of the total population and may result from a few matings.
3. The second aim was to test the hypothesis that the variation among pools within a stream was greater after dry periods than after wet periods. This was because after wet periods, larvae would have greater opportunity for mixing because of movement among pools. This hypothesis was partly supported by the mitochondrial DNA data but not by the allozyme data, in which variation among pools was extremely low on both sampling occasions. The reasons for this difference are unclear.     

Cloning,expression and characterization of a eukaryotic cycloalkanone monooxygenase from <Emphasis Type="Italic">Cylindrocarpon radicicola</Emphasis> ATCC 11011

In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer–Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.     

4-Methylumbelliferyl esters as fluorogenic substrates for proteases

4-Methylumbelliferyl esters of amino acid derivatives have been synthesized using the carbodiimide, disulphite and carbonate methods. Of these, the first was shown capable of preparing 2-naphthyl and 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonyl glycine and benzyloxycarbonyl-citrulline but not of benzoyl-N^G-nitroarginine. 2-Naphthyl benzoyl-N^G-nitroargininate was prepared successfully using di(2-naphthyl)sulphite. Bis(4-methylumbelliferyl)sulphite could not be prepared but 4-methylumbelliferyl benzoyl-N^G-nitroargininate was obtained by the use of an equilibrium method using diphenyl sulphite in the presence of 4-methylumbelliferone. A new reagent, phenyl 4-methylumbelliferyl carbonate, was synthesized and used for the preparation of the 4-methylumbelliferyl esters of benzoylglycine, benzyloxycarbonylglycine and benzoyl-N^G-nitroarginine. The 4-methylumbelliferyl esters of benzyloxycarbonylglycine and benzyloxycarbonylcitrulline were shown to be good substrates for the assay of proteases, including chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4). Disadvantages of 4-methylumbelliferyl esters are discussed.     

Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.