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), an enzyme of the amino sugar utilization pathway, has been purified from an overproducing strain of Escherichia coli. The enzyme is a tetramer of identical 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 belonging to the alpha/beta structural family. In the native enzyme, two thiols per chain are titrated with 5-5'-dithio-bis(2-nitrobenzoate) (NbS2); one reacts rapidly, the other more slowly. The reaction of the more reactive sulfhydryl completely inhibits the activity of the enzyme. Three thiols, of the total of eight per subunit of the native enzyme, are modified by methyl iodide without significantly changing the kinetic parameters; the methylated enzyme becomes insensitive to NbS2 inhibition. One of the enzyme reaction products, glucosamine 6-phosphate, completely protects this thiol from NbS2 reaction. The kinetics of the deacetylase reaction have been studied both in the forward direction and in the backward direction. The reverse reaction is strongly unfavored and is probably physiologically insignificant, but it was useful for obtaining a better kinetic description of the enzyme. A sequential mechanism, with ordered release of products and a slow isomerization of the enzyme-acetate complex, is proposed. This model is supported by data from substrate and product inhibition patterns in both directions of the reaction.
Souza
J M
JM
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City D.F., Mexico.
Plumbridge
J A
JA
Calcagno
M L
ML
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry
D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D000581:Amidohydrolases; C018717:N-acetylglucosamine-6-phosphate deacetylase