Use of synthetic substrates to study binding and catalysis by lysozyme

Rand-Meir, Tsafrira; Dahlquist, Frederick W.; Raftery, Michael A.

doi:10.1021/bi00838a044

Cited by 63 publications

(27 citation statements)

References 22 publications

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“…Inhibitory activities of Nacetylglucosamine (NAG), N,N'-diacetylglucosamine (NAG-NAG: N,N'-diacetylchitobiose), and N,N',NЉ,N'Љ-tetraacetylglucosamine (NAG-NAG-NAG-NAG: N,N',NЉ,NЉ'-tetraacetylchitotetraose) were examined (Sigma Corporation Canada). These are structurally similar to TAC but have a different number of NAG units (8). Two somewhat related sugars also were examined to see if there was potential for blocking the activity of lysozyme.…”

Section: In Vitro Experimentsmentioning

confidence: 99%

“…Once the nature of this myocardial depressant factor was identified, we showed in the right ventricular trabecular preparation that purified Lzm-S from canine spleen could be blocked by a competitive inhibitor of lysozyme N,N',NЉ-triacetylglucosamine, also referred to as N,N',NЉ-triacetylchitotriose (TAC) or chitotriose (5,8). TAC is the trisaccharide of NAG that has three NAG residues linked by ␤ (1-4) glycosidic linkages and competitively inhibits the enzymatic activity of Lzm-S.…”

mentioning

confidence: 99%

“…This would provide additional evidence that the carbohydrate portion of the cardiac membrane glycoprotein was important in Lzm-S's myocardial depressant effect. Furthermore, we tested a series of NAG oligosaccharides and variants to the NAG structure as inhibitors of Lzm-S's depressant activity in the right ventricular trabecular preparation (8,13).…”

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confidence: 99%

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N,N′,N′-triacetylglucosamine, an inhibitor of lysozyme, prevents myocardial depression in Escherichia coli sepsis in dogs*

Mink

Jacobs

Duke

et al. 2004

Critical Care Medicine

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Section: In Vitro Experimentsmentioning

confidence: 99%

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confidence: 99%

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N,N′,N′-triacetylglucosamine, an inhibitor of lysozyme, prevents myocardial depression in Escherichia coli sepsis in dogs*

Mink

Jacobs

Duke

et al. 2004

Critical Care Medicine

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“…2b) To reduce the rate of the second step in a different approach, we incorporated¯uorine in place of the 2-acetamido group of chitobiose, while keeping a good leaving group at the anomeric centre. Previous studies, designed to rule out a possible mechanism involving anchimeric assistance from the 2-acetamido group, demonstrated that substrates bearing hydrogen and hydroxyl substitutions at this centre are hydrolysed at similar rates to that of the parent compound bearing the acetamido group, and thus reveal that the amide functionality is not critical for catalysis 14,15 . An electronegative¯uorine substituent at the 2 position will, however, inductively destabilize the oxocarbenium ion-like transition states leading to the formation and hydrolysis of the intermediate, slowing both steps.…”

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confidence: 99%

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Vocadlo

Davies

Laine

et al. 2001

Nature

596

456

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Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

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“…Because lysozyme has been suggested to distort the pyranose ring of the substrate bound in site D (see Section 11), Giudici and Bruice have examined the hydrolysis of methyl-a-~-2,6-anhydroaltropyranoside (48) in an effort to ascertain whether distortion of the sub-H-0 0-CH, 48 strate to achieve coplanarity of the C-2, C-1, 0, and C-5 atoms is suf€icient to bring about a change of mechanism from A-1 to rate limiting protonation ( 102). Both dichloroacetic acid and formic acid were ineffective at enhancing the rate of hydrolysis.…”

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confidence: 99%