Biomedical subjects

B Svensson

Publications and source records attributed to B Svensson.

At least 109 records · Page 6Linked to original sources

PubMed1997

Radiographic osteoarthritis of the knee classified by the Ahlbäck and Kellgren & Lawrence systems for the tibiofemoral joint in people aged 35-54 years with chronic knee pain.

OBJECTIVES: To determine the prevalence of tibiofemoral radiographic knee osteoarthritis (OA) in people aged 35-54 years associated with chronic (> 3 months) knee pain using two different radiographic grading systems. METHODS: Population based postal survey in a random sample of inhabitants in a district in southern Sweden followed by clinical examination and plain posteroanterior, weight bearing radiographical examination. The Ahlbäck criteria (focusing on joint space narrowing) and the Kellgren & Lawrence classification for knee OA were used for diagnosing tibiofemoral OA. RESULTS: A questionnaire was sent to 2000 randomly selected people aged 35-54 years. The response rate was 92.6%. Fifteen per cent of these people reported chronic knee pain. This group (n = 279) was offered a clinical and radiographic examination of the knee joint and 204 persons agreed to participate. According to the Kellgren & Lawrence classification 28 subjects had OA of the knee grade 2 or more and 16 grade 3 or more. Radiographically detected OA of the knee according to Ahlbäck was found in 20 cases. The minimum prevalence of radiological tibiofemoral knee OA with knee pain was thus 1.5% for Kellgren & Lawrence grade 2 or more, 0.9% for grade 3 or more, and 1.1% according to the Ahlbäck classification. The agreement between the Kellgren & Lawrence grades 2-3 versus Ahlbäck grade I as well as grade 3-4 versus Ahlbäck grade I-II was good (kappa 0.76 and 0.78 respectively). CONCLUSION: The prevalence of radiographic tibiofemoral OA combined with chronic knee pain in people aged 35-54 years was around 1% as estimated by either the Kellgren & Lawrence or the Ahlbäck classifications systems. Prospective follow up of this cohort should elucidate the significance of knee pain as a sign of developing OA.

Adult↗

PubMed1996

Substrate binding mechanism of Glu180-->Gln, Asp176-->Asn, and wild-type glucoamylases from Aspergillus niger.

Glucoamylase (1,4-alpha-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus, of which the 3D structure is known, releases beta-D-glucose from the non-reducing ends of starch and other related oligo and polysaccharides, cleaving the alpha-1,4-bond positioned between subsites 1 and 2 in the enzyme-substrate complex. The presteady and steady state kinetics of two of the existing mutants, Glu180-->Gln and Asp176-->Asn, are presented here. The kinetic results are analyzed according to two reaction models: One suggested previously [Olsen, K., Svensson, B., & Christensen, U. (1992) Eur. J. Biochem. 209, 777-784], which contains three consecutive steps of the reaction, and one generally accepted and used in calculations of subsite energies [Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6], which assumes important non-productive binding and identical values of the intrinsic catalytic constant independent of the chain length of the substrate. It is found that glucoamylase shows kinetics in accordance with a consecutive three-step mechanism, in which the formation of the Michaelis complex occurs in two steps and is followed by a slow catalytic step and fast dissociation of the products with no accumulation of enzyme-product complexes. The kinetics, however, are not in accordance with the model generally used in subsite energy calculations. Thus the kinetic model on which very low values of subsite 1 and high values of subsite 2 interaction energies have been based is not correct. A greater importance of subsite 1 interactions than has hitherto been anticipated is indicated. The results of the Glu180-->Gln mutant show weak overall binding, which stems from large effects on the formation of the Michaelis complex in the second step of the reaction, but no or rather small effects on the initial association of enzyme and substrate, except for maltose. The mutant further shows effective catalysis. A hydrogen bond of the side chain carboxylate of Glu180 with the 2-OH of the sugar ring at subsite 2 is an expected important interaction of the Michaelis complex, as seen from the 3D structures of stabile enzyme-inhibitor complexes. Apparently this bond is established in the second reaction step. It is indicated that subsite 1 and 3 interactions to a great extent govern the initial association. In accordance with a dynamic role of Glu180, structural energy minimization calculations show a flexibility of the gamma-carboxylate of Glu180. The side chain of Asp176 participates in a hydrogen-bonding network also involving the backbone of Glu180 and Glu179, the catalytic acid. Compared with the wild-type enzyme, the Asp176-->Asn mutant shows no significant changes in binding. The catalytic rate is, however, markedly reduced. Apparently changes in the hydrogen bonding network of Asp176 are of importance in the rate-determining catalytic step, but not in the substrate binding steps. Structural energy minimization calculations on the Asp176-->Asn mutant, however, do not confirm this assumption.

Asparagine↗

PubMed1996

A model for the photosystem II reaction center core including the structure of the primary donor P680.

For a detailed understanding of the function of photosystem II (PSII), a molecular structure is needed. The crystal structure has not yet been determined, but the PSII reaction center proteins D1 and D2 show homology with the L and M subunits of the photosynthetic reaction center from purple bacteria. We have modeled important parts of the D1 and D2 proteins on the basis of the crystallographic structure of the reaction center from Rhodopseudomonas viridis. The model contains the central core of the PSII reaction center, including the protein regions for the transmembrane helices B, C, D, and E and loops B-C and C-D connecting the helices. In the model, four chlorophylls, two pheophytins, and the nonheme Fe2+ ion are included. We have applied techniques from computational chemistry that incorporate statistical data on side-chain rotameric states from known protein structure and that describe interactions within the model using an empirical potential energy function. The conformation of chlorophyll pigments in the model was optimized by using exciton interaction calculations in combination with potential energy calculations to find a solution that agrees with experimentally determined exciton interaction energies. The model is analyzed and compared with experimental results for the regions of P680, the redox active pheophytin, the acceptor side Fe2+, and the tyrosyl radicals TyrD and TyrZ. P680 is proposed to be a weakly coupled chlorophyll a pair which makes three hydrogen bonds with residues on the D1 and D2 proteins. In the model the redox-active pheophytin is hydrogen bonded to D1-Glu130 and possibly also to D1-Tyr126 and D1-Tyr147. TyrD is hydrogen bonded to D2-His190 and also interacts with D2-Gln165. TyrZ is bound in a hydrophilic environment which is partially constituted by D1-Gln165, D1-Asp170, D1-Glu189, and D1-His190. These polar residues are most likely involved in proton transfer from oxidized TyrZ or in metal binding.

Amino Acid Sequence↗

PubMed1996

Structure and energetics of the glucoamylase-isomaltose transition-state complex probed by using modeling and deoxygenated substrates coupled with site-directed mutagenesis.

Molecular recognition, site-directed mutagenesis, and molecular modeling are combined to describe hydrogen bonds important for formation and catalysis of the Aspergillus niger glucomylase-isomaltose complex. This analysis of the energetics of the transition-state complex identified OH-4', -6', and -4 as critical for isomaltose hydrolysis. Side-chains hydrogen bonded to isomaltose OH-4 (reducing end unit, i.e. at glucoamylase binding subsite 2) induced substrate conformation adjustment to optimize binding energy contributed by charged hydrogen bonds to OH-4' and -6' at the non-reducing unit (i.e. at subsite 1). These interactions were evident in the modeled complex of glucoamylase and isomaltose in the preferred trans-gauche conformation. Kinetic analysis demonstrated reductions in kcat of 10(3) to 10(5)-fold for the corresponding deoxy- and O-methyl analogs of isomaltose. Analysis of two mutants at the level of subsite 2, Glu 180-->Gln and Asp 309-->Glu, showed the binding energy for the enzyme-transition state complex, delta delta G, contributed by OH-3 and -4 to be 6-7 kJ mol-1 weaker than with wild-type enzyme. Unexpectedly, however, substitution of isomaltose OH-4' and -6' (at subsite 1) resulted in 10 to 12 kJ mol-1 lower delta delta G++ for both the mutants. Mutation at subsite 2, therefore, strongly perturbed distant transition-state stabilizing interactions. This was confirmed with 4'- and 6'-deoxy analogs of the conformationally biased methyl 6-R-C-methyl-alpha-isomaltoside, readily adopting trans-gauche conformation, that exhibit full delta delta G++ 18 to 20 kJ mol-1 for both mutants and wild-typ-. Glucoamylase, during catalysis, thus seems to induce a change from the predominant solution gauche-gauche conformer to trans-gauche isomaltose. This leads to enhanced binding at subsite 1 in the enzyme transition-state complex.

Aspergillus niger↗

PubMed1996

Primary structure of the plant serpin BSZ7 having the capacity of chymotrypsin inhibition.

The primary structure of barley grain serpin BSZ7 was deduced from a cDNA encoding 397 amino-acid residues. More than 70% of the residues were confirmed by sequencing peptide fragments. The N-terminus was identified as an acetylated Ala by using mass spectrometry coupled with amino-acid analysis. None of the four putative N-glycosylation sites were found to be glycosylated. The positional identity of BSZ7 with plant and mammalian serpins is 69-72% and 25-32%, respectively.

Amino Acid Sequence↗

PubMed1996

Mutational modulation of substrate bond-type specificity and thermostability of glucoamylase from Aspergillus awamori by replacement with short homologue active site sequences and thiol/disulfide engineering.

Rational protein engineering based on three-dimensional structure, sequence alignment, and previous mutational analysis served to increase thermostability and modulate bond-type specificity in glucoamylase from Aspergillus awamori. The single free cysteine, Cys320, became disulfide bonded in the Ala246 --> Cys mutant, thus enhancing T50 by 4 degrees C to 73 degrees C. Compared to wild-type, Ala246 --> Cys was roughly twice as active at 66 degrees C, but half as active at 45 degrees C. The alternative, elimination of the thiol group in Cys320 --> Ala, barely improved thermostability or altered activity. Secondly, to acquire exceptionally high specificity toward alpha-1,6 glucosidic linkages, characteristic of Hormoconis resinae glucoamylase, two short sequential mutants, Val181 --> Thr/Asn182 --> Tyr/Gly183 --> Ala(L3 glucoamylase) and Pro307 --> Ala/Thr310 --> Val/Tyr312 --> Met/Asn313 --> Gly (L5 glucoamylase), were made. These homologue mutants are located in the (alpha/alpha)6-fold of the catalytic domain in segments that connect alpha-helices 5 and 6 and alpha-helices 9 and 10, respectively. The kinetics of malto- and isomaltooligosaccharides hydrolysis clearly demonstrated that combination of the mutations in L3L5 compensated adverse effects of the single replacements in L3 or L5 glucoamylases to yield wild-type or higher activity. On alpha-1,4-linked substrates, typically Km increased 2-fold for L3, and Kcat decreased up to 15-fold for L5 glucoamylase. In contrast, on alpha-1,6-linked substrates L3 showed both a 2-fold increase in Km and a 3-fold decrease in kcat, while L5 GA caused a similar kcat reduction, but up to 9-fold increase in Km. L3L5 glucoamylase had remarkably low Km for isomaltotriose through isomaltoheptaose and elevated kcat on isomaltose, resulting in an approximately 2-fold improved catalytic efficiency (kcat/Km). Rational loop replacement thus proved powerful in achieving variants with enhanced properties of a highly evolved enzyme.

Amino Acid Sequence↗

PubMed1996

Crystallographic complexes of glucoamylase with maltooligosaccharide analogs: relationship of stereochemical distortions at the nonreducing end to the catalytic mechanism.

Crystal structures at pH 4 of complexes of glucoamylase from Aspergillus awamori var. X100 with the pseudotetrasaccharides D-gluco-dihydroacarbose and acarbose have been refined to R-factors of 0.147 and 0.131 against data to 1.7- and 2.0-A resolution, respectively. The two inhibitors bind in nearly identical manners, each exhibiting a dual binding mode with respect to the location of the last sugar residues. The reduced affinity of D-gluco-dihydroacarbose (K1 = 10(-8) M) relative to acarbose (K1 = 10(-12) M) may stem in part from the weakening of hydrogen bonds of the catalytic water (Wat 500) to the enzyme. Steric contacts between the nonreducing end of D-gluco-dihydroacarbose and the catalytic water perturb Wat 500 from its site of optimal hydrogen bonding to the active site. Interactions within the active site displace the 6-hydroxymethyl group of the nonreducing end of both acarbose and D-gluco-dihydroacarbose toward a more axial position. In the case of D-gluco-dihydroacarbose the shift in the position of the 6-hydroxymethyl group occurs with a 12 degrees change in two dihedral angles of the glucopyranose ring toward a half-chair conformation. The observed conformational distortion of the first residue of D-gluco-dihydroacarbose is consistent with the generation of a glucopyranosyl cation in the transition state. Comparable distortions of stereochemistry in model compounds require approximately 2 kcal/mol, not more than 25% of the energy necessary to form the half-chair conformation in glucose. The magnitude of stereochemical distortion observed in the active site of glucoamylase suggests that favorable electrostatic interactions between the putative glucopyranosyl cation intermediate and the active site must be more important in stabilizing the transition state than mechanical distortion of the substrate.

Acarbose↗

PubMed1996

Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis.

Synthesis of heme A from heme B (protoheme IX) most likely occurs in two steps with heme O as an intermediate. Bacillus subtilis CtaB, an integral membrane protein, functions in farnesylation of heme B to form heme O. CtaA, also a membrane protein, is required for heme A synthesis from heme O and appears to be a monooxygenase and/or a dehydrogenase. Wild-type ctaA and ctaB expressed together from plasmids in B. subtilis resulted in CtaA containing equimolar amounts of low-spin heme B and heme A; this form of CtaA was named cyt ba-CTA. A mutant ctaB gene was identified and characterised. It encodes a truncated CtaB polypeptide. Wild-type ctaA and the mutant ctaB gene on plasmids resulted in CtaA containing mainly low-spin heme B; this variant was named cyt b-CTA. The heme B component in cyt ba-CTA and cyt b-CTA showed identical properties; a mid-point redox potential of +85 mV, an EPR g(max) signal at 3.7, and a split alpha-band light absorption peak. The heme A component in cyt ba-CTA showed a mid-point potential of +242 mV, an EPR g(max) signal at 3.5, and the alpha-band light absorption peak at 585 nm. It is suggested that the CtaA protein contains two heme binding sites, one for heme B and one for substrate heme. The heme B would play a role in electron transfer, i.e. function as a cytochrome, in the monooxygenase and/or dehydrogenase reaction catalysed by CtaA whereas heme O/heme A would be substrate/product.

Bacillus subtilis↗

PubMed1996

Catalytic mechanism of glucoamylase probed by mutagenesis in conjunction with hydrolysis of alpha-D-glucopyranosyl fluoride and maltooligosaccharides.

The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using alpha-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116-->Ala, Trp120-->Phe, Asp176-->Asn, and Glu400-->Gln. These replacements decreased the activity (kcat/KM) toward maltose by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GAs, showed wild-type and twice wild-type level activity, while Asp176-->Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild-type GA complexed with GF, the pH-activity (kcat) profile shows a pKa of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis.

Aspergillus niger↗

PubMed1996

A circularly permuted alpha-amylase-type alpha/beta-barrel structure in glucan-synthesizing glucosyltransferases.

A motif of amino acid residues, located at the active site and specific beta-strands in alpha-amylases, is recognized in alpha-1,3- and alpha-1,6-glucan-synthesizing glucosyltransferases, leading to the conclusion that these enzymes contain an alpha/beta-barrel closely related to the (beta/alpha)8-fold of the alpha-amylase superfamily. The secondary structure elements of the transferase barrel, however, are circularly permuted to start with an alpha-helix equivalent to helix 3 in the alpha-amylases. Thus, the transferase counterpart of the long third beta-->alpha connection--constituting a domain in the alpha-amylases--is divided to precede and succeed the barrel. This architectural arrangement may be coupled to sucrose scission and glucosyl transfer. The involvement in the mechanism in glucosyltransferases of active site residues recurring in amylolytic enzymes is discussed.

Amino Acid Sequence↗

PubMed1996

Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.

Amylases↗

PubMed1996

Development of community nursing: analysis of the central services and practice dilemmas.

As examples of nurse practitioners in primary care, Swedish district nurses have been shown, in several studies, to respond well to the general pressure to achieve higher productivity in health care. Yet they have less frequently been involved in studies of the detailed content of the service they provide. To maintain total service quality, change should not be brought about only for the sake of efficacy. This study is divided into an analysis of the basic service provided in district nursing by identifying a typical interaction with a patient; and an analysis of the daily dilemmas district nurses experience, their consequences, and possible measures for change. For the consultation study, data were collected from 40 videorecordings of office-hour consultations by a rural district nurse. Regarding the dilemma situations, a critical incident questionnaire was returned by 153 district nurses in a Swedish country. The analyses showed that the district nurse practises in an area between self-care on the one hand, and the intersection of the different specialties in health care, on the other. Both the consultation study and the study of dilemmas in practice showed that the district nurse has to approach the health care organisation from a viewpoint close to that of the patient, which implies that she can, and has to, maintain an overview of the patient's total interaction with social institutions. This focal localisation of the shared care and co-ordination aspects constitutes a critical target for change and development efforts. The conclusions are that, first, the role of the district nurse/nurse practitioner as co-ordinator and advocate in patient-centred care could be considered in central health services policies and planning. Second, the content of the district nurses' daily work could be taken into account in the implementation of quality programs. These measures together can lead to an integration between today's theoretical nursing models and the dynamically changing structures of health care organisations.

Adult↗

PubMed1996

Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: powerful analytical tools in recombinant protein chemistry.

Electrospray ionization and matrix assisted laser desorption/ionization are effective ionization methods for mass spectrometry of biomolecules. Here we describe the capabilities of these methods for peptide and protein characterization in biotechnology. An integrated analytical strategy is presented encompassing protein characterization prior to and after cloning of the corresponding gene.

Biotechnology↗

PubMed1995

Chemoenzymatic synthesis of 6 omega-S-alpha-D-glucopyranosyl-6 omega-thiomaltooligosaccharides: their binding to Aspergillus niger glucoamylase G1 and its starch-binding domain.

A coupling reaction of cyclodextrin glucosyltransferase (CGTase) with glucose and 6-deoxy-6-iodo-cyclomaltoheptaose (1), in the presence of glucoamylase, followed by acetylation, led to a convenient synthesis of acetylated 6III-deoxy-6III-iodo-maltotriose (2) and 6IV-deoxy-6IV-iodomaltotraose (3). Nucleophilic displacement of the iodine atom of these protected maltotriose and maltotetraose analogs by the activated form of 2,3,4,6-tetra-O-acetyl-1-S- acetyl-1-thio-alpha-D-glucose (4) afforded peracetylated 6III-S-alpha-D-glucopyranosyl-6III-thiomaltotriose (5) and 6IV-S-alpha-D-glucopyranosyl-6IV-thiomaltotetraose (6) in high yield. The interaction of OH-free tetra- and penta-saccharides (7 and 8) with both glucoamylase G1 from Aspergillus niger as well as its isolated starch-binding domain fragment were studied by UV difference spectroscopy. It was found that the starch-binding domain has higher affinity for 7 and 8 than for maltotetraose and maltopentaose.

Aspergillus niger↗

PubMed1995

Increased levels of mitogen activated protein kinase (MAP-K) detected in the injured adult mouse sciatic nerve.

Adult mouse sciatic nerves (SNs) with attached dorsal root ganglia (DRG) were analysed for the presence of mitogen activated protein kinase (MAP-K) during normal and regenerative conditions. By immunohistochemistry, MAP-K was found to be present in the normal nerve at low levels in both Schwann cells and DRG nerve cell bodies, with a profoundly increased expression during regeneration. In axonal outgrowth assays, treatment with 2 mM 2-aminopurine (2-AP), a MAP-K antagonist, inhibited the regeneration of axons from the SN as well as from the cultured superior cervical ganglia. The reduced outgrowth was probably not due to toxic effects of the drug since the ganglionic protein synthesis was not inhibited. It is possible that 2-AP interferes with regeneration-related events by inhibition of MAP-K.

2-Aminopurine↗

PubMed1995

Thermodynamics of inhibitor binding to mutant forms of glucoamylase from Aspergillus niger determined by isothermal titration calorimetry.

We have investigated the binding of mutant forms of glucoamylase from Aspergillus niger to the inhibitors 1-deoxynojirimycin and acarbose. The mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each mutant we have determined both the affinities for the two inhibitors and the thermodynamic state functions for binding using titration microcalorimetry. We find that acarbose binds to all the mutants with a wide range of binding constants (10(4) < Ka < 10(13) M-1). In contrast, 1-deoxynojirimycin shows either binding at near wild-type affinity (Ka approximately equal to 10(4) M-1) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the known three-dimensional structure of the wild-type enzyme with subsites 1, 2, and 3 being important for acarbose binding while only subsite 1 is critical for 1-deoxynojirimycin binding. In most of the mutants studied the magnitudes of the enthalpies and the entropies of binding of the mutant enzymes differed from those of the wild-type enzyme with the mutant enzymes having a relatively large portion of their binding energy composed of enthalpy and a relatively small proportion composed of entropy. The pattern of changes in the enthalpy and entropy is hypothesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.

1-Deoxynojirimycin↗

PubMed1995

Mutational analysis of the roles in catalysis and substrate recognition of arginines 54 and 305, aspartic acid 309, and tryptophan 317 located at subsites 1 and 2 in glucoamylase from Aspergillus niger.

The mutants Arg54-->Leu, Arg54-->Lys, Arg305-->Lys, Asp309-->Glu, and Trp317-->Phe, located at subsites 1 and 2 in glucoamylase from Aspergillus niger, provide insight into the importance of specific hydrogen bonds and hydrophobic interactions in substrate recognition, catalytic mechanism, and stability. As suggested from the crystal structure of a closely related glucoamylase [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639], Arg54 in subsite 1 hydrogen bonds to the key polar group 4'-OH of maltose. The two mutants of Arg54 display losses in transition-state stabilization of 16-21 kJ mol-1 in the hydrolysis of different maltooligodextrins, which originate from a [(1.2-1.8) x 10(3)]-fold reduction in kcat and changes in Km ranging from 25% to 300% of the wild-type values. Arg305 similarly hydrogen bonds to 2'-OH and 3-OH, located at subsites 1 and 2, respectively. Arg305-->Lys glucoamylase is not saturated at concentrations of maltose or maltoheptaose of 400- and 40-fold, respectively, the Km of the wild-type enzyme. This mutant also has highly reduced kcat. On the other hand, for the alpha-1,6-linked isomaltose, the Lys305 mutant surprisingly has the same Km as the wild-type enzyme, while kcat is 10(3)-fold reduced. Arg305 is thus an important determinant in the distinction of the alpha-1,4 to alpha-1,6 substrate specificity. Arg305 interacts electrostatically and hydrophobically with the side chains of Asp309 and Trp317.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗

PubMed1995

Arg-27, Arg-127 and Arg-155 in the beta-trefoil protein barley alpha-amylase/subtilisin inhibitor are interface residues in the complex with barley alpha-amylase 2.

Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents. Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2-complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI. Since Arg-27 is not conserved in rice and wheat ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155].

Amino Acid Sequence↗