On the role of the N-terminal group in the allosteric function of glucosamine-6-phosphate deaminase from Escherichia coli

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) from Escherichia coli is an allosteric enzyme of the K-type, activated by N-acetylglucosamine 6-phosph ate. It is a homohexamer and has six allosteric sites located in clefts bet ween the subunits. The amino acid side-chains in the allosteric site involv ed 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-enz ymic transamination reaction, and we further reduced the product with boroh ydride, to obtain the corresponding enzyme with a terminal hydroxy group. S everal experimental controls were performed to assess the procedure, includ ing reconditioning of the enzyme samples by refolding chromatography. Allos teric activation by N-acetylglucosamine 6-phosphate became of the K-V mixed type in the transaminated protein. Its kinetic study suggests that the all osteric equilibrium for this modified enzyme is displaced to the R state, w ith the consequent loss of co-operativity. The deaminase with a terminal hy droxy acid, obtained by reducing the transaminated enzyme, showed significa nt recovery of the catalytic activity and its allosteric activation pattern became similar to that found for the unmodified enzyme. It had lost, howev er, 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 g roup plays a part in the co-operativity of the enzyme and, more importantly , indicate that the loss of this cooperativity at low pH is due to the hydr onation of this amino group. (C) 2000 Academic Press.