The active sites of fructose 6-phosphate,2-kinase: Fructose-2,6- bisphosphatase from rat testis. Roles of ASP-128, THR-52, THR-130, ASN-73, and TYR-197

To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate,2-kinase:fructose- 2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr- 197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in V(max) and a significant increase in K(m) values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high K(m) for both substrates and an 800-fold decreased V(max). Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in K(Fru) (6-P) with only a small increase in K(ATP) and small changes in V(max). Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased V(max). A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with K(m)= 0.64 μM, and V(max) = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The K(d) values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.