Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs.

Boel, Esper; Hjort, I; Svensson, Birte; Norris, Fanny; Norris, Kjeld; Fiil, Niels P.

doi:10.1002/j.1460-2075.1984.tb01935.x

Cited by 175 publications

(79 citation statements)

References 38 publications

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“…10% of non-ligand binding enzyme molecules which were eliminated by affinity chromatography on acarbose-Sepharose (section 3.2). The tryplic fragments of G2 deriv- in the amino acid sequence ofglucoamylase (5,30). The UV-absorption of the isolated peptide (Figure 3) confirmed the presence of an intact tryptophan in the fragment from G2 oxidized in the presence of acarbose, while in contrast the fragment from the inactive G2 derivative exhibited oxindolealanine UV-absorption.…”

Section: Resultsmentioning

confidence: 83%

“…100 amino acid residues, bul the two forms have essentially the same activity on soluble substrates (31). The amino acid sequence of the enzyme (5,6,30) near the Abbreviations: AH = aminohexyl; G1 and G2 = the larger and the smaller of the two forms ofglucoamylase from A. niger (31); HPLC = high pressure liquid chromatography; NBS = N-bromosuecinimide; TFA = trifluoroacetic acid; Tris = 2-amin~-2(hydroxymethyl)-l, 3-propandioI. essential residue resembles that near a tryptophanyl residue of Taka-amylase A, proposed to interact with substrate in the active site cleft of this a-amylase (20,33). O.Oz Fine Chemicals, Uppsala, Sweden.…”

Section: Introductionmentioning

confidence: 99%

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Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Clarke

Svensson

1984

Carlsberg Res. Commun.

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Enzymatically active, N-bromosuccinimide oxidized glucoamylase G2 (EC 3.2.1.3) was prepared in the presence of the inhibitor acarbose and inactive, oxidized G2 with capacity to bind substrate was prepared in the presence of maltose. Four out of 15 tryptophanyl residues were oxidized in the first derivative and five in the latter. In order to identify this fifth and essential residue, tryptie fragments of the two G2 derivatives were isolated. The peptide fragment from the inactive G2 der/vative containing the additional oxindolealaniae residue was Tocated in HPLC chromatograms by UV-absorption measurements. Normal and second derivative UV-spectra, amino acid composition and NH_,-terminal sequence of the isolated fragment led to identification of Trp(120) as a residue essential for activity. A short homologous amino acid sequence precedes both this Trp(120) and the Trp(83) which is involved in substrate binding in Taka-amylase A (J. Biochem. 95, 697-702 (1984)).

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Section: Resultsmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Clarke

Svensson

1984

Carlsberg Res. Commun.

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“…(30) and A. saitoi (27) in the presence of substrates or inhibitors has previously led to the suggestion that the essential carboxyl groups are located at or near subsites 2 and 3. Crystals suitable for structural analysis have not been obtained from a glucoamylase, but substantial sequence homology exists (29,53) between the enzymes from A. niger (6,50,51), Rh. oryzae (4), S. cerevisiae (diastaticus) (60), Saccharomycopsis fibuligera (29) and S. cerevisiae (59).…”

Section: Introductionmentioning

confidence: 99%

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Svensson

Møller

Clarke

1988

Carlsberg Res. Commun.

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Glucoamylases GI and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC). A tolal of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the GI and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pK~ 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these fig,ands, differenlial labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC.Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of K~ and a slight reduction of V,~ for hydrolysis of starch relative to the unmodified enzyme. Both K~ and Vm~ for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC.In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

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“…X I 0 0 has not been cloned and we are unable to make specific mutant forms. However, the corresponding gene from A. awamori [24], which is identical to the gene isolated from A. niger [15], is available for molecular engineering. We are therefore able to compare the NMR spectra of glucoamylases from both of these with mutant forms.…”

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confidence: 99%

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

Firsov¹,

Neustroev²,

Aleshin

et al. 1994

European Journal of Biochemistry

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'H-NMR spectra have been recorded for glucoamylases I and I1 from Aspergillus awamori var. X I 0 0 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 D,O. 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 D,O. 8-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 Cel and C82 protons of an uncharged imidazole ring. We identify D as arising from the Ne2 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 Trpl20. 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 11, 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.

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Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs.

Cited by 175 publications

References 38 publications

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range

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