Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from <i>Aspergillus awamori</i>

Sierks, Michael R.; Ford, Clark; Reilly, Peter J.; Svensson, Birte

doi:10.1093/protein/3.3.193

Cited by 132 publications

(122 citation statements)

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“…Glu179 and Glu400 have been identified as the general acid and the general base catalyst, respectively, and the pH dependencies of steady-state kinetic parameters are in accordance with a rate-determining hydrolysis involving these two catalytic residues Sierks et al, 1990;Harris et al, 1993;Frandsen et al, 1994). Also in accordance with this, the mutation of Glu400 to Gln results in a reduction of k, to 3%, showing the marked influence of this residue on the rate-determining step (Frandsen et al, 1996).…”

supporting

confidence: 56%

“…The three-dimensional structures of the native enzyme as well as of tight-binding inhibitor-enzyme complexes are known (Aleshin et al, 1992(Aleshin et al, , 1994a(Aleshin et al, , b, 1996Harris et al, 1993;Stoffer et al, 1995). Two carboxyl groups, those of Glu179 and Glu400, have been assigned, respectively, as general acid and general base catalysts in the rate-determining hydrolytic reaction step, a result based on pH dependencies of kinetic parameters (Sierks et al, 1990;Frandsen et al, 1994), chemical modification studies and structural data (Harris et a]., 1993). The general reaction scheme (Model I) of glucoamylase-catalyzed reactions based on previous stopped-flow kinetic results involves three reactions steps: (a) formation of a first enzyme-substrate association complex (ES); (b) change of conformation of the first enzyme-substrate complex to form the Michaelis complex (E:") and (c) the rate-determining, actual catalysis (Olsen et al, 1992(Olsen et al, , 1993Christensen et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

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Some Details of the Reaction Mechanism of Glucoamylase from Aspergillus Niger— Kinetic and Structural Studies on Trp52→Phe and Trp317→Phe Mutants

Christensen

Stoffer

Svensson

et al. 1997

European Journal of Biochemistry

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Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases TrpS24Phe and Trp3 17+Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trpl20-Phe and Glul80+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 (p-flank). The Trp317-Phe and the Glul80--.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 Arg30S 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. TrpS2 and Trpl20 each interact with the catalytic acid, Glu179, and are placed on the flank (a-flank) of the active site opposite to Trp317, Arg30S and Glu180. Also the TrpS2-Phe and T r p l 2 b P h e 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 P-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg30S and Glu180 of the /$flank are established and the correct alignment on the a-flank of Glu179, the general acid catalyst, governed by its flexible interactions with TrpS2 and Trp120, occurs.Keywords: glucoamylase; mutation of TrpS2 and of Trp317 ; presteady-state kinetics ; binding-reaction models.Glucoamylase (1,4-a-D-glUCan glucohydrolase) is an inverting exoglycosidase, producing P-D-glUCoSe by hydrolysis of a-1,4-glucosidic bonds (and, with much less efficiency, n-I ,6-bonds) from the non-reducing end of starch and related oligoand polysaccharides (Hiromi et al., 1966a(Hiromi et al., , b, 1983. Glucoamylase from Aspergilli exists in two natural forms: G l and G2. The GI form froin Aspergillus niger (Alal -Arg616) consists of a catalytic and a starch-binding domain connected with a highly 0-glycosylated linker region. The G2 form (Alal -ProS12) lacks the starch-binding domain and the ability to degrade raw starch. The amino acid sequence of the G2 form is identical to Alal-Pro512 of the G1 form (Sven...

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supporting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Some Details of the Reaction Mechanism of Glucoamylase from Aspergillus Niger— Kinetic and Structural Studies on Trp52→Phe and Trp317→Phe Mutants

Christensen

Stoffer

Svensson

et al. 1997

European Journal of Biochemistry

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“…The carboxylate group of the side chain of El79 has been identified as a probable acid catalyst essential for enzymic activity [22,44,451. One of its oxygen atoms is hydrogenbonded to three different side chain groups: the indole NH groups of WS2 and W120 and an amide hydrogen of Q124.…”

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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“…Chemical Rescue of the Inactive E294A Mutant-Although the substantial decrease in the catalytic activity of the mutant enzyme lacking the putative nucleophilic residue is consistent with its suggested role, the identification of the catalytic residues could not be based on the reduction in activity alone. There are several examples where single mutations in proposed catalytic residues of glycoside hydrolases resulted in reduced or undetectable activity (46,47), whereas crystallographic and biochemical analyses showed that these residues are not involved directly in catalysis (48,49). To unambiguously identify Glu-294 as the nucleophilic residue of AbfA T-6, the azide rescue methodology was used.…”

Section: Figmentioning

confidence: 99%

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

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Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Cited by 132 publications

References 0 publications

Some Details of the Reaction Mechanism of Glucoamylase from Aspergillus Niger— Kinetic and Structural Studies on Trp52→Phe and Trp317→Phe Mutants

Some Details of the Reaction Mechanism of Glucoamylase from Aspergillus Niger— Kinetic and Structural Studies on Trp52→Phe and Trp317→Phe Mutants

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

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

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