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 W alker motif residues of rat testis fructose 6-phosphate,2-kinase:fruct ose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constr ucted 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 resulte d in a small decrease in V-max and a significant increase in K-m value s for both substrates, These mutants exhibited similar pH activity pro files as that of the wild type enzyme, Mutation of Thr-52 to Ala resul ted in an enzyme with an infinitely high K-m for both substrates and a n 800-fold decreased V-max Substitution of Asn-73 with Ala or Asp caus ed a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in K-ATP and small changes in V-max. Mutation of Thr-13 0 caused small changes in the kinetic properties, Replacement of Tyr-1 97 with Ser resulted in an enzyme with severely decreased binding of F ru 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 mu M, and V-max = 25 milli-units/mg and was a competitive inh ibitor with respect to ATP, When mant-ATP bound to the enzyme, fluores cence intensity at 440 nm increased, mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method, T he K-d values of the T52A and D128N enzymes were infinitely high and c ould not be measured, while those of the other mutant enzymes increase d slightly, These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crysta llographic 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-kin ase reaction is mediated via a transition state stabilization mechanis m.