Richard Hall - N-Acetyl-D-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

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      Publication Details (including relevant citation   information):

      Hall, Richard S.; Xiang, Dao Feng; Xu, Chengfu; Raushel, Frank   M..  N-Acetyl-D-glucosamine-6-phosphate Deacetylase:   Substrate Activation via a Single Divalent Metal   Ion.    Biochemistry  (2007),    46(27),  7942-7952.  CODEN: BICHAW    ISSN:0006-2960.  CAN 147:252820  AN   2007:635391    CAPLUS

      Abstract:

      NagA is a member of the amidohydrolase superfamily and catalyzes   the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The   catalytic mechanism of this enzyme was addressed by the   characterization of the catalytic properties of metal-substituted   derivatives of NagA from Escherichia coli with   a variety of substrate analogues. The reaction mechanism is of   interest since NagA from bacterial sources is found with either   one or two divalent metal ions in the active site. This   observation indicates that there has been a divergence in the   evolution of NagA and suggests that there are fundamental   differences in the mechanistic details for substrate activation   and hydrolysis. NagA from E. coli was inactivated by the   removal of the zinc bound to the active site and the apoenzyme   reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+,   Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the   reaction is initiated by the polarization of the carbonyl group   of the substrate via a direct interaction with the divalent metal   ion and His-143. The invariant aspartate (Asp-273) found at the   end of beta-strand 8 in all members of the amidohydrolase   superfamily abstracts a proton from the metal-bound water   molecule (or hydroxide) to promote the hydrolytic attack on the   carbonyl group of the substrate. A tetrahedral intermediate is   formed and then collapses with cleavage of the C-N bond after   proton transfer to the leaving group amine by Asp-273. The lack   of a solvent isotope effect by D2O and the absence of any changes   to the kinetic constants with increases in solvent viscosity   indicate that net product formation is not limited to any   significant extent by proton-transfer steps or the release of   products. N-Trifluoroacetyl-d-glucosamine-6-phosphate is   hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl   derivative. This result is consistent with the formation or   collapse of the tetrahedral intermediate as the rate limiting   step in the catalytic mechanism of NagA.

      Address (URL): http://pubs.acs.org/cgi-bin/abstract.cgi/bichaw/2007/46/i27/abs/bi700543x.html