Glucosamine-6-phosphate deaminase (EC 22.214.171.124) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosphate. It is a homohexamer and has six allosteric sites located in clefts between the subunits. The amino acid side-chains in the allosteric site involved in phosphate binding are Arg158, Lys160 and Ser151 from one subunit and the N-terminal amino group from the facing polypeptide chain. To study the functional role of the terminal amino group, we utilized a specific non-enzymic transamination reaction, and we further reduced the product with borohydride, to obtain the corresponding enzyme with a terminal hydroxy group. Several experimental controls were performed to assess the procedure, including reconditioning of the enzyme samples by refolding chromatography. Allosteric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein. Its kinetic study suggests that the allosteric equilibrium for this modified enzyme is displaced to the R state, with the consequent loss of co-operativity. The deaminase with a terminal hydroxy acid, obtained by reducing the transaminated enzyme, showed significant recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme. It had lost, however, the pH-dependence of homotropic co-operativity shown by the unmodified deaminase in the pH range 6-8. These results show that the terminal amino group plays a part in the co-operativity of the enzyme and, more importantly, indicate that the loss of this co- operativity at low pH is due to the hydronation of this amino group.
Copyright 2000 Academic Press.