Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1

Dorfmueller, H.C.; Fang, W.; Rao, F.V.; Blair, D.E.; Attrill, H.; van Aalten, D.M.F.

doi:10.1107/S0907444912019592

Figure 3
(a) Apparent K_m of WT and C141S CeGNA1 for both substrates. The velocity (µM s⁻¹) is shown as a function of the concentration of the second substrate. The WT enzyme has approximately twofold higher affinity for both substrates than the active-site mutant C141S CeGNA1. The catalytic efficiency of the mutant has dropped to around 20% (Table 2

). However, the covalent adduct shows no detectable signal above the background (Table 2

). (b) Double-reciprocal plots of initial rates of formation of CoA catalysed by CeGNA1. In this plot, the concentration of acetyl-CoA is varied from 50 to 500 µM at several fixed concentrations of GlcN-6P. (c) pH optima of WT and C141S CeGNA1. To obtain the bell-shaped curves the nonlinear fit was calculated with GraFit. WT and C141S CeGNA1 have the same pH optimum at pH 8.2. (d) Mass spectrometry supports the covalent bond between the active-site Cys141 and the product CoA in the crystal structure (space group P6₁). 15 min incubation (at 293 K) with 5 mM DTT was sufficient to release the covalently bonded CoA, corresponding to the mass of one released CoA molecule (767 Da). (e) Activity of the CeGNA1–Cys–CoA adduct dimer compared with CeGNA1 in solution after incubation with various concentrations of DTT as indicated. Incubation of 10 nM CeGNA1–Cys–CoA without DTT does not give any detectable activity above the background. Reactivation of 98.2% can be achieved with 5 mM DTT for 15 min at 293 K.

BIOLOGICAL
CRYSTALLOGRAPHY

ISSN: 1399-0047

Volume 68| Part 8| July 2012| Pages 1019-1029

doi:10.1107/S0907444912019592

Search term		doi		Advanced search
Author		volume	page