burst kinetics | ScienceGate

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

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Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as ‘burst’ kinetics on fluorescent polymeric model substrates

Biochemical Journal ◽

10.1042/bj20041144 ◽

2005 ◽

Vol 385 (2) ◽

pp. 527-535 ◽

Cited By ~ 84

Author(s):

Kalle KIPPER ◽

Priit VÄLJAMÄE ◽

Gunnar JOHANSSON

Keyword(s):

Bacterial Cellulose ◽

Trichoderma Reesei ◽

Anthranilic Acid ◽

Degree Of Polymerization ◽

Cellulose Substrates ◽

Quantitative Monitoring ◽

End Groups ◽

Alternative Means ◽

Burst Kinetics ◽

Hydrolysis Of

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

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Tyr-503 of β-galactosidase (Escherichia coli) plays an important role in degalactosylation

Biochemistry and Cell Biology ◽

10.1139/o99-042 ◽

1999 ◽

Vol 77 (3) ◽

pp. 229-236 ◽

Cited By ~ 9

Author(s):

Robert M Penner ◽

Nathan J Roth ◽

Beatrice Rob ◽

Helga Lay ◽

Reuben E Huber

Keyword(s):

Escherichia Coli ◽

Acid Catalysis ◽

Covalent Bond ◽

Reaction Rates ◽

Amino Acid Residues ◽

Wild Type ◽

Burst Kinetics ◽

Inhibition Studies ◽

Ks Values ◽

Lines Of Evidence

Substitutions for Tyr-503 of β-galactosidase caused large decreases of the activity. Both the galactosylation (k2) and degalactosylation (k3) rates were decreased. Substitutions by residues without transferable protons, caused k3 to decrease much more than k2 while substitutions with residues having transferable protons, caused approximately equal decreases of k2 and k3. Several lines of evidence showed this. The Km values of the substituted enzymes were much smaller than those for the wild type if the substituted amino acid residues did not have transferable protons; this was not the case when the substituted residues had transferable protons. Inhibition studies showed that the Km values were not small because of small Ks values but were small because of relatively small k3 values (compared with the k2 values). The conclusion that the k3 values are small relative to k2 upon substitution with residues without transferable protons is also based upon other studies: studies indicating that the reaction rates were similar with different substrates, studies in the presence of alcohol acceptors, studies showing that the rate of inactivation by 2,4-dinitrophenyl-2-deoxy-2-F-β-D-galactopyranoside decreased much less than the rate of reactivation; studies on burst kinetics, and pH studies. The data suggest that Tyr-503 may be important for the degalactosylation reaction because of its ability to transfer protons and thereby facilitate cleavage of the transient covalent bond between galactose and Glu-537. Key words: β-galactosidase, tyrosine, mechanism, acid catalysis.

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