N-ACETYLGLUCOSAMINE-6-PHOSPHATE DEACETYLASE FROM ESCHERICHIA-COLI - PURIFICATION AND MOLECULAR AND KINETIC CHARACTERIZATION

N-Acetylglucosamine-6-phosphate deacetylase (E.C.3.5.1.25), and enzyme of the amino sugar utilization pathway, has been purified form an ove rproducing strain of Escherichia coli. The enzyme is a tetramer of ide ntical 41-kDa subunits. The sedimentation coefficient of the oligomer is 6.5 s(20,w) and it has a pI of 4.9. The circular dichroism spectrum of the enzyme in the far uv range suggests that it is a protein belon ging to the alpha/beta structural family. In the native enzyme, two th iols per chain are titrated with 5,5'-dithio-bis(2-nitrobenzoate) (NbS 2);(4) one reacts rapidly, the other more slowly. The reaction of the more reactive sulfhydryl completely inhibits the activity of the enzym e. Three thiols, of the total of eight per subunit of the native enzym e, are modified by methyl iodide without significantly changing the ki netic parameters; the methylated enzyme becomes insensitive to NbS2 in hibition. One of the enzyme reaction products, glucosamine 6-phosphate , completely protects this thiol from NbS2 reaction. The kinetics of t he deacetylase reaction have been studied both in the forward directio n and in the backward direction. The reverse reaction is strongly unfa vored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequentia l mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported b y data from substrate and product inhibition patterns in both directio ns of the reaction. (C) 1997 Academic Press.