Side chain reactivities of glucoamylase G2 from aspergillus niger evaluated by group-specific chemical modifications

Håkansson, Kjell; Svensson, Birte

doi:10.1007/bf02907184

Cited by 6 publications

(3 citation statements)

References 47 publications

(57 reference statements)

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“…Hikansson and Svensson [32] showed that treatment of GIIn with diethylpyrocarbonate or iodoacetamide led to modification of only one histidine. In the reaction with iodacetamide only H254 was alkylated as shown by amino acid sequence analysis of the derivatized enzyme.…”

Section: Discussionmentioning

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have provided additional information.Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined.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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Section: Discussionmentioning

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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“…However, peptide mapping of Cys400 GA treated with iodo[2-3 H]acetic acid and MALDI MS of the radiolabeled peptide revealed alkylation of only His254 in approximately 50% yield. This residue is far from the active site of GA (18), and was previously shown to undergo carboxymethylation without affecting activity (36). Accordingly, no significant overalkylation was detected by MALDI MS analysis since the wild-type (25), Cys400 GA, and Cys400 GA treated with iodoacetic acid all gave a very broad peak (reflecting the glucan heterogeneity of GA) centered around a mass of 82.3 kDa.…”

Section: Resultsmentioning

confidence: 89%

“…These reactions resulted in only minor activity increases and partial oxidation of Cys400 as determined by peptide mapping coupled with MALDI MS analysis (data not shown). These reactions with KI alone could not be controlled, and after a transient gain in activity, inactivation dominated which was probably caused by iodination of aromatic residues (36). Hence, conditions were established to gently oxidize Cys400 GA in a manner analogous to the treatment of the enzyme with 4-bromobutyric acid which had led to 160% activity relative to wildtype GA.…”

Section: Resultsmentioning

confidence: 99%

Restoration of Catalytic Activity beyond Wild-Type Level in Glucoamylase from Aspergillus awamori by Oxidation of the Glu400→Cys Catalytic-Base Mutant to Cysteinesulfinic Acid

et al. 1998

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Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

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Herden

1992

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Side chain reactivities of glucoamylase G2 from aspergillus niger evaluated by group-specific chemical modifications

Cited by 6 publications

References 47 publications

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

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

Restoration of Catalytic Activity beyond Wild-Type Level in Glucoamylase from Aspergillus awamori by Oxidation of the Glu400→Cys Catalytic-Base Mutant to Cysteinesulfinic Acid

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