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PubMed1983

Stability of enzymes in urine at 37 degrees C.

The activity of alanine aminopeptidase, alkaline phosphatase, gamma-glutamyltransferase, lactate dehydrogenase and beta-N-acetyl-D-glucosaminidase in urine at 37 degrees C was investigated by a model simulating in vivo conditions. The stability of these urinary enzymes is influenced particularly by pH. At low pH values in urine (about pH 5.0) the four first-mentioned enzymes rapidly lose a considerable part of their activity, whereas beta-N-acetyl-D-glucosaminidase is inactivated at higher pH values in urine (about at pH 8.0). This inactivation effect is also time-dependent and can be modified by urinary substances such as creatinine, urea and electrolytes. To avoid misinterpretation of enzyme activity determinations in urine, the simultaneous measurement of urinary pH should be performed.

Creatinine↗
PubMed2000

N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes.

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

Amino Acid Sequence↗
PubMed2003

Enzyme reactor design under thermal inactivation.

Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme-catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes, and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

Bioreactors↗
PubMed2005

Characterization of human SCD2, an oligomeric desaturase with improved stability and enzyme activity by cross-linking in intact cells.

SCD (stearoyl-CoA desaturase) catalyses the conversion of saturated fatty acids into mono-unsaturated fatty acids, a critical step involved in lipid metabolism and various other biological functions. In the present study, we report the identification and characterization of a human gene that encodes a novel SCD enzyme (hSCD2). The hSCD2 gene codes for a 37.5-kDa protein that shares 61% and 57% sequence identity with the human SCD1 and mouse SCD2 enzymes respectively. The recombinant hSCD2 enzyme expressed in mammalian and Sf9 insect cells efficiently catalysed desaturation of both stearoyl- and palmitoyl-CoAs to the corresponding mono-unsaturated fatty acids. In comparison with the hSCD1 gene that is predominantly expressed in liver, hSCD2 is most abundantly expressed in pancreas and brain. Additionally, hSCD2 transcripts from adult and foetal tissues exhibit different sizes because of alternative splicing in the non-coding region, suggesting that hSCD2 expression is developmentally regulated. The recombinant human SCD2 and SCD1 transiently expressed in COS-7 cells exhibited as oligomeric proteins that consist of homodimers and oligomers when resolved by SDS/PAGE. The complex formation was independent of SCD protein expression levels, as supported by a relatively constant ratio of the level of dimers and oligomers to that of the monomers from COS-7 cells transiently transfected with different amounts of SCD expression vectors. Furthermore, treatment of intact COS-7 cells with a cross-linking reagent resulted in dose-dependent increases in the levels of SCD protein and activity, suggesting that oligomerization may play an important role in regulating the stability of SCD enzymes.

Amino Acid Sequence↗
PubMed1998

Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans.

Xylanases from family 10 glycanases contain three conserved histidine residues in their active site. The role of H86 in the structure-function of xylanase A from Streptomyces lividans (XlnA) was studied by site-directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produced, purified and characterized. The six mutations reduced the affinity of XlnA towards xylan without having any major effect on the catalytic constant. All these mutations also lowered the pKa of the acid-base catalyst by 0.46-1.94 pH units. The mutations decreased the enzyme stability at 60 degrees C by up to 95% and the transition temperature by 2.2-5.8 degrees C. Unfolding of the protein with guanidine hydrochloride (GdnxHCl) showed that five out of six mutations decreased the concentration required to denature 50% of the XlnA, confirming the importance of H86 for the stability of the enzyme. The increase in m value ¿m=d(deltaG)/d[GdnxHCl]¿ also suggested the involvement of residue H86 in the structure of the denatured state of XlnA. It can be concluded from this study that this active site residue was conserved in family 10 glycanases for its function in maintaining the elevated pKa of the acid-base catalyst and in the stability of the protein, while being of little importance for the activity.

Binding Sites↗
PubMed2006

Chemical modification of protein surfaces to improve their reversible enzyme immobilization on ionic exchangers.

The enzyme penicillin G acylase (PGA) is not adsorbed at pH 7 on DEAE- or PEI-coated supports, neither is it adsorbed on carboxymethyl (CM)- or dextran sulfate (DS)-coated supports. The surface of the enzyme was chemically modified under controlled conditions: chemical amination of the protein surface of carboxylic groups (using soluble carbodiimide and ethylendiamine) and chemical succinylation (using succinic anhydride) of amino groups. The full chemical modification produced some negative effects on enzyme stability and activity, although partial modification (mainly succinylation) presented negligible effects on both enzyme features. The chemical amination of the protein surface permitted the immobilization of the enzyme on CM- and DS-coated support, while the chemical succinylation permitted the enzyme immobilization on DEAE- and PEI-coated supports. Immobilization was very strong on these supports, mainly in the polymeric ones, and dependent on the degree of modification, although the enzymes still can be desorbed after inactivation by incubation under drastic conditions. Moreover, the immobilization on ionic polymeric beds allowed a significant increase in enzyme stability against the inactivation and inhibitory effects of organic solvents, very likely by the promotion of a certain partition of the organic solvent out of the enzyme environment. These results suggest that the enrichment of the surface of proteins with ionic groups may be a good strategy to take advantage of the immobilization of industrial enzymes via ionic exchange on ionic polymeric beds.

Adsorption↗
PubMed2006

The natural mutation by deletion of Lys9 in the thrombin A-chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding.

The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (deltaK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (alpha-NAPAP) to the wild-type (WT) and deltaK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free deltaK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the deltaK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A-B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.

Catalytic Domain↗
PubMed1988

Properties of a highly purified mitochondrial deoxyguanosine kinase.

Deoxyguanosine kinase, purified over 6000-fold from beef liver mitochondria by means of deoxyguanosine-3'-(4-aminophenyl phosphate)-Sepharose affinity chromatography, was nearly homogeneous. It phosphorylates only deoxyguanosine and deoxyinosine among the natural nucleosides, with apparent Km values of 4.7 and 21 microM, respectively. Among nucleoside analogs tested, only arabinosylguanine (Ki = 125 microM) and 8-aza-deoxyguanosine (Ki = 450 microM) competed with deoxyguanosine. The relative molecular mass of the enzyme is 56,000, as determined by equilibrium sedimentation, and sodium dodecyl sulfate-gel electrophoresis suggests two subunits of Mr 28,000. The pH optimum for enzyme activity is 5.5, but optimum enzyme stability is seen at pH 7.0. Triton X-100 increased the stability of the enzyme markedly. ATP is the best phosphate donor at pH 5.5, but pyrimidine triphosphates such as dTTP and UTP are more efficient donors at pH 7.4. The activation energy, at pH 5.5, was estimated to be 10.9 kcal/mol. Amino acid modification experiments suggest the involvement of arginine, cysteine, and probably histidine. The inactivation of the enzyme by modification of these amino acid residues was time and pH dependent. Both substrates protected the enzyme from inactivation in every case but that of photooxidation by Rose Bengal, where only deoxyguanosine prevented inactivation.

Animals↗
PubMed1984

Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions.

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.

Acetylcholinesterase↗
PubMed2005

Impact of reaction conditions on the laccase-catalyzed conversion of bisphenol A.

The oxidative conversion of aqueous BPA catalyzed by laccase from Trametes versicolor was conducted in a closed, temperature-controlled system containing buffer for pH control. The effects of medium pH, buffer concentration, temperature and mediators and the impacts of dissolved wastewater constituents on BPA conversion were investigated. The optimal pH for BPA conversion was approximately 5, with greater than half maximal conversion and good enzyme stability in the range of 4-7. The stability of the enzyme was not impacted by buffer concentration, nor was BPA conversion. Despite the observation that the enzyme tended to be inactivated at elevated temperatures, enhanced conversion of BPA was observed up until a reaction temperature of 45 degrees C. Of the mediators studied, ABTS was most successful at enhancing the conversion of BPA. Dissolved wastewater constituents that were studied included various inorganic salts, organic compounds and heavy metal ions. BPA conversion was inhibited in the presence of anions such as sulfite, thiosulfate, sulfide, nitrite and cyanide. The metal ions Fe(III) and Cu(II) and the halogens chloride and fluoride substantially suppressed BPA conversion, but the presence of selected organic compounds did not significantly reduce the conversion of BPA.

Anions↗
PubMed2002

Electron transfer between yeast cytochrome bc(1) complex and cytochrome c: a structural analysis.

The structure of the complex between cytochrome c (CYC) and the cytochrome bc(1) complex (QCR) from yeast crystallized with an antibody fragment has been recently determined at 2.97 A resolution [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2800]. CYC binds to subunit cytochrome c(1) of the enzyme stabilized by hydrophobic interactions surrounding the heme crevices creating a small, compact contact site. A central cation-pi interaction is an important and conserved feature of CYC binding. Peripheral patches with highly conserved complementary charges further stabilize the enzyme-substrate complex by long-range electrostatic forces and may affect the orientation of the substrate. Size and characteristics of the contact site are optimal for a transient electron transfer complex. Kinetic data show a bell-shaped ionic strength dependence of the cytochrome c reduction with a maximum activity near physiological ionic strength. The dependence is less pronounced in yeast compared to horse heart CYC indicating less impact of electrostatic interactions in the yeast system. Interestingly, a local QCR activity minimum is found for both substrates at 120-140 mM ionic strength. The architecture of the complex results in close distance of both c-type heme groups allowing the rapid reduction of cytochrome c by QCR via direct heme-to-heme electron transfer. Remarkably, CYC binds only to one of the two possible binding sites of the homodimeric complex and binding appears to be coordinated with the presence of ubiquinone at the Q(i) site. Regulatory aspects of CYC reduction are discussed.

Crystallization↗
PubMed2003

Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme.

The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein. The separation from AMP deaminase of an 85-kDa component with a blocked N terminus is observed when the enzyme is applied to the Zn-charged column under nondenaturing conditions. On storage under reducing conditions, this component undergoes an 85- to 95-kDa transition yielding a L-T-P-T-D-X-K-T-T-K-P-L N-terminal sequence, suggesting that the shift in the migration on SDS/PAGE as well as the truncation of the protein at its N terminus are promoted by the reduction of a disulfide bond present in freshly isolated HPRG. The separation of HPRG induces a marked reduction in the solubility of AMP deaminase, strongly suggesting a role of HPRG in assuring the molecular integrity of the enzyme.

AMP Deaminase↗
PubMed1978

Microbial metabolism of amino alcohols. Formation of coenzyme B12-dependent ethanolamine ammonia-lyase and its concerted induction in Escherichia coli.

1. Kinetic studies of ethanolamine ammonia-lyase formation by Escherichia coli suggested that coenzyme B12 (5'-deoxyadenosylcobalamin), with ethanolamine, is a co-inducer. 2. Enzymic and immunological tests failed to show the formation of complementary enzyme components induced separately by ethanolamine and cobalamin respectively. 3. Although specific for ethanolamine as the substrate, enzyme formation was induced by certain analogues, e.g. 2-aminopropan-1-ol. 4. Experiments with cyano[57Co]-cobalamin suggested that neither coenzyme B12 nor some more tightly bound coenzymically inactive cobamide was necessary for enzyme stability in vitro. 5. Mutants of E. coli were obtained which formed ethanolamine ammonia-lyase apoenzyme constitutively, showing that neither ethanolamine nor cobalamin was required for assembly or post-transcriptional stability of the enzyme in vivo. Constitutive enzyme formation was subject to catabolite repression, particularly by glucose. 6. It appears likely that ethanolamine and coenzyme B12, acting in concert, induce ethanolamine ammonia-lyase formation. The term 'concerted' induction is proposed for this phenomenon.

Ammonia-Lyases↗
PubMed1998

Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin.

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.

Alkylation↗
PubMed2000

Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase.

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 +/- 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2. 00 +/- 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 +/- 0.07 and the copper to 0.97 +/- 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper (</=0.02 mol/mol of NOS). Most (> or =75%) of the bound zinc was released from baculovirus-expressed rat nNOS by p-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 +/- 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 +/- 0.05 mol of zinc but had a specific activity of 426 +/- 46 nmol of citrulline.mg(-1).min(-1), corresponding to 93 +/- 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H(4)biopterin). H(4)biopterin did not affect zinc release. Even in the presence of H(4)biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.

4-Chloromercuribenzenesulfonate↗