Dataset Information

Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

ABSTRACT: Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5'-phosphate-dependent enzyme catalyzing CO(2) release from the alpha-carboxyl group of L-glutamate to yield gamma-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5'-phosphate-Lys(276) Schiff base with the distal nitrogen of His(465), the penultimate residue in the GadB sequence. Herein we show that His(465) has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His(465) contributes with n approximately 2.5 to the overall cooperativity of the system. The residual cooperativity (n approximately 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His(465), dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His(465), a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His(465) mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.

SUBMITTER: Pennacchietti E

PROVIDER: S-EPMC2797229 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

Pennacchietti Eugenia E Lammens Tijs M TM Capitani Guido G Franssen Maurice C R MC John Robert A RA Bossa Francesco F De Biase Daniela D

The Journal of biological chemistry 20090921 46

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5'-phosphate-dependent enzyme catalyzing CO(2) release from the alpha-carboxyl group of L-glutamate to yield gamma-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the ...[more]

PMID: 19797049

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Dataset Information

Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

Publications

Mutation of His465 alters the pH-dependent spectroscopic properties of Escherichia coli glutamate decarboxylase and broadens the range of its activity toward more alkaline pH.

Similar Datasets

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