RESUMEN
Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure-function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X-ray structures have been determined at 1.9 A resolution for [G175Q]HBP and at 2.5 A resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2 % and 20.7 %, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS-induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS.
Asunto(s)
Aprotinina/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Lípido A/metabolismo , Lipopolisacáridos/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos , Sitios de Unión , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Cristalización , Glicoproteínas/química , Humanos , Interleucina-6/metabolismo , Isótopos de Yodo , Lípido A/química , Lipopolisacáridos/química , Monocitos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Difracción de Rayos XRESUMEN
Transferred nuclear Overhauser effect (trNOE) experiments have been performed to investigate the conformations of the competitive inhibitors, methyl 5'-thio-4-N-alpha-maltoside 3a and methyl 5'-thio-4-S-alpha-maltoside 4 when bound to the catalytic subunit of the enzyme glucoamylase. These NMR data suggest that, although each of the free ligands populates two conformational families, both heteroanalogues are bound by the enzyme in conformations in the area of the global energy minimum. These conformations have been used as initial points for docking into the active site of the enzyme taken from a X-ray crystal structure of the related glucoamylase-D-gluco-dihydroacarbose 2 complex. Minimization of the resulting complexes has yielded structures for the bound complexes. Corroboration of the structures is provided by fast T(1)(rho)-relaxation effects for certain ligand protons as a result of close contacts with protons in the enzyme active site. The results auger well for the combined use of transferred NOE spectroscopy and molecular modeling based on X-ray crystal structures of complexes of suitable congeners for the rapid analysis of ligand-receptor interactions.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Glicósidos/química , Acarbosa/farmacología , Sitios de Unión , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética/métodos , Metilglucósidos/farmacología , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
The synthesis of a series of 5-thio-D-glucopyranosylarylamines by reaction of 5-thio-D-glucopyranose pentaacetate with the corresponding arylamine and mercuric chloride catalyst is reported. The products were obtained as anomeric mixtures of the tetraacetates which can be separated and crystallized. The tetraacetates were deprotected to give alpha/beta mixtures of the parent compounds which were evaluated as inhibitors of the hydrolysis of maltose by glucoamylase G2 (GA). A transferred NOE NMR experiment with an alpha/beta mixture of 7 in the presence of GA showed that only the alpha isomer is bound by the enzyme. The Ki values, calculated on the basis of specific binding of the alpha isomers, are 0.47 mM for p-methoxy-N-phenyl-5-thio-D-glucopyranosylamine (7), 0.78 mM for N-phenyl-5-thio-D-glucopyranosylamine (8), 0.27 mM for p-nitro-N-phenyl-5-thio-D-glucopyranosylamine (9) and 0.87 mM for p-trifluoromethyl-N-phenyl-5-thio-D-glucopyranosylamine (10), and the K(m) values for the substrates maltose and p-nitrophenyl alpha-D-glucopyranoside are 1.2 and 3.7 mM, respectively. Methyl 4-amino-4-deoxy-4-N-(5'-thio-alpha-D-glucopyranosyl)-alpha-D-glucopyrano side (11) is a competitive inhibitor of GA wild-type (Ki 4 microM) and the active site mutant Trp120-->Phe GA (Ki 0.12 mM). Compounds 7, 8, and 11 are also competitive inhibitors of alpha-glucosidase from brewer's yeast, with Ki values of 1.05 mM, > 10 mM, and 0.5 mM, respectively. Molecular modeling of the inhibitors in the catalytic site of GA was used to probe the ligand-enzyme complementary interactions and to offer insight into the differences in inhibitory potencies of the ligands.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Glucosamina/análogos & derivados , Glicósido Hidrolasas/antagonistas & inhibidores , Compuestos de Sulfhidrilo/síntesis química , Sitios de Unión , Secuencia de Carbohidratos , Inhibidores Enzimáticos/farmacología , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Glucosamina/síntesis química , Inhibidores de Glicósido Hidrolasas , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica , Saccharomyces cerevisiaeRESUMEN
To mimic the structure of the 1.8-fold more active (k(cat)) Rhizopus oryzae glucoamylase (GA), Aspergillus niger GA was subjected to site-directed mutagenesis in the Trp170-Tyr175 segment of the third of the six well-conserved alpha-->alpha connecting loops of the catalytic (alpha/alpha)6-barrel. While the Trp170-->Phe, Gln172-->Asn and Tyr175-->Phe mutants showed an up to 1.7-fold increased k(cat) and Gly174-->Cys GA and approximately 2-fold reduced k(cat) towards maltotriose and longer substrates, Asn171-->Ser, Thr173-->Gly and A.niger wild-type GA had very similar kcat and K(m) values for the hydrolysis of isomaltose and the malto-oligosaccharides of DP 2-7. Crystal structures of pseudotetrasaccharide inhibitor complexes of Aspergillus awamori var. X100 GA, which is 94% identical to A.niger GA, indicate that Tyr175 is located at binding subsite 4, while the preceding target residues and the high-mannose type unit on Asn171 are at a larger distance from the site of catalysis. The mutations had a modest effect on thermostability; the temperature for 50% inactivation, Tm, was thus unchanged for Tyr175 -->Phe GA and reduced by 0.2-2.9 degrees C for the other mutants. The deletion of the N-linked high-mannose unit-in Asn171 -->Ser and Thr173-->Gly GAs-appeared to be of minor importance for enzyme activity and thermostability, and did not increase the sensitivity to proteolysis.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/genética , Mutagénesis Sitio-Dirigida/fisiología , Rhizopus/enzimología , Secuencia de Aminoácidos , Aspergillus niger/genética , Secuencia de Bases , Sitios de Unión/genética , Catálisis , Secuencia Conservada , Estabilidad de Enzimas/genética , Glucano 1,4-alfa-Glucosidasa/química , Datos de Secuencia Molecular , Estructura Molecular , Ingeniería de Proteínas , Rhizopus/genética , Relación Estructura-ActividadRESUMEN
Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/metabolismo , Sitios de Unión , Catálisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Fluorescencia , Glucano 1,4-alfa-Glucosidasa/genética , Enlace de Hidrógeno , Cinética , Mutagénesis Sitio-Dirigida/genética , Unión Proteica , Conformación Proteica , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/genética , Eritrocitos/enzimología , Modelos Moleculares , Mutación , Piruvato Quinasa/química , Secuencia de Aminoácidos , Niño , Humanos , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismoRESUMEN
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.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Glucano 1,4-alfa-Glucosidasa/genética , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Cinética , Modelos Químicos , Mutación Puntual , Unión Proteica , TermodinámicaRESUMEN
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.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/química , Isomaltosa/química , Modelos Moleculares , Secuencia de Carbohidratos , Disacáridos/química , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Enlace de Hidrógeno , Isomaltosa/análogos & derivados , Isomaltosa/metabolismo , Cinética , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/química , Conformación ProteicaRESUMEN
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.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Estabilidad de Enzimas , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/genética , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligosacáridos/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/química , Glucano 1,4-alfa-Glucosidasa/química , Oligosacáridos/química , Trisacáridos/química , Acarbosa , Aspergillus/enzimología , Sitios de Unión , Secuencia de Carbohidratos , Catálisis , Simulación por Computador , Cristalografía por Rayos X , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Modelos Moleculares , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
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.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , 1-Desoxinojirimicina/metabolismo , Acarbosa , Sitios de Unión , Calorimetría/métodos , Secuencia de Carbohidratos , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Termodinámica , Trisacáridos/metabolismoRESUMEN
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)
Asunto(s)
Arginina/metabolismo , Ácido Aspártico/metabolismo , Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Triptófano/metabolismo , Arginina/genética , Ácido Aspártico/genética , Secuencia de Bases , Catálisis , Análisis Mutacional de ADN , Cartilla de ADN , Estabilidad de Enzimas , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/genética , Guanidina , Guanidinas , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Especificidad por Sustrato , Termodinámica , Triptófano/genéticaRESUMEN
The crystal structure at pH 4 of the complex of glucoamylase II(471) from Aspergillus awamori var. X100 with the pseudotetrasaccharide D-gluco-dihydroacarbose has been refined to an R-factor of 0.125 against data to 2.2 A resolution. The first two residues of the inhibitor bind at a position nearly identical to those of the closely related inhibitor acarbose in its complex with glucoamylase at pH 6. However, the electron density bifurcates beyond the second residue of the D-gluco-dihydroacarbose molecule, placing the third and fourth residues together at two positions in the active site. The position of relatively low density (estimated occupancy of 35%) corresponds to the location of the third and fourth residues of acarbose in its complex with glucoamylase at pH 6. The position of high density (65% occupancy) corresponds to a new binding mode of an extended inhibitor to the active site of glucoamylase. Presented are possible causes for the binding of D-gluco-dihydroacarbose in two conformations at the active site of glucoamylase at pH 4.
Asunto(s)
Aspergillus/enzimología , Glucano 1,4-alfa-Glucosidasa/química , Conformación Proteica , Trisacáridos/química , Sitios de Unión , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Glucano 1,4-alfa-Glucosidasa/metabolismo , Estructura Molecular , Trisacáridos/metabolismoRESUMEN
Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrates ground-state binding and transition-state stabilization. Compared to those of the wild-type enzyme, Km values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400-->Gln mutant, with kcat values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48-->Trp A. niger glucoamylase kcat is reduced 80-100-fold, while Km is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48-->Trp and Glu400-->Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function- and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aspergillus niger/genética , Glucano 1,4-alfa-Glucosidasa/genética , Ácido Glutámico/genética , Glutamina/genética , Mutagénesis Sitio-Dirigida , Tirosina/genética , Aspergillus niger/metabolismo , Secuencia de Bases , Ácidos Carboxílicos/metabolismo , Catálisis , Cartilla de ADN , Estabilidad de Enzimas , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Enlace de Hidrógeno , Datos de Secuencia Molecular , Termodinámica , Tirosina/metabolismoRESUMEN
1H-NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. delta-D-gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 protons of an uncharged imidazole ring. We identify D as arising from the N epsilon 2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information. Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered. The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Aspergillus/enzimología , Sitios de Unión , Glucano 1,4-alfa-Glucosidasa/antagonistas & inhibidores , Glucano 1,4-alfa-Glucosidasa/genética , Espectroscopía de Resonancia Magnética , Mutación , Conformación Proteica , ProtonesRESUMEN
The catalytic domain of glucoamylases G1 and G2 from Aspergillus niger is produced in vitro in high yield by limited proteolysis using either subtilisin Novo or subtilisin Carlsberg. Purification by affinity chromatography on an acarbose-Sepharose column followed by ion-exchange chromatography on HiLoad Q-Sepharose leads to separation of a number of structurally closely related forms of domain. The cleavage occurs primarily between Val-470 and Ala-471 as indicated by C-terminal sequencing, whereas the N-terminus is intact. Subtilisin Carlsberg, in addition, produces a type of domain which is hydrolysed before Ser-444, an O-glycosylated residue. This leaves the fragment Ser-444-Val-470 disulphide-bonded to the large N-terminal part of the catalytic domain. Subtilisin Novo, in contrast, tends to yield a minor fraction of forms extending approx. 30-40 amino-acid residues beyond Val-470. The thermostability is essentially the same for the single-chain catalytic domain and the original glucoamylases G1 and G2, whereas the catalytic domain cut between Ser-443 and Ser-444 is less thermostable. For both types of domain the kinetic parameters, Km and kcat., for hydrolysis of maltose are very close to the values found for glucoamylases G1 and G2.
Asunto(s)
Aspergillus niger/enzimología , Glucano 1,4-alfa-Glucosidasa/biosíntesis , Secuencia de Aminoácidos , Catálisis , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Glucano 1,4-alfa-Glucosidasa/aislamiento & purificación , Glucano 1,4-alfa-Glucosidasa/metabolismo , Calor , Hidrólisis , Datos de Secuencia MolecularRESUMEN
For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (greater than 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ alkylation after electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.