TYR254 HYDROXYL GROUP ACTS AS A 2-WAY SWITCH MECHANISM IN THE COUPLING OF HETEROTROPIC AND HOMOTROPIC EFFECTS IN ESCHERICHIA-COLI GLUCOSAMINE-6-PHOSPHATE DEAMINASE

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was fi rst proposed on the basis of a theoretical analysis of the sequence an d demonstrated by spectrophotometric experiments. Two tyrosine residue s, Tyr121 and Tyr254, were indicated as involved in the mechanism of c ooperativity and in the allosteric regulation of the enzyme [Altamiran o et al. (1994) fur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T-->R t ransition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been s hown to be part of the allosteric pocket [Oliva et al. (1995) Structur e 3, 1323-1332]. Although it is not directly involved in binding the a llosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydro gen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotro pic effects. Homotropic cooperativity diminished and the allosteric ac tivation pattern changed from one of the K-type in the wild-type deami nase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase i s kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the int eractions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosami ne 6-P deaminase.