In the study of allosteric proteins, understanding which effector-protein interactions contribute to allosteric activation is important both for designing allosteric drugs and for understanding allosteric mechanisms. The antihyperglycemic target, human liver pyruvate kinase (hL-PYK), binds its allosteric activator, fructose 1,6-bisphosphate (Fru-1,6-BP), such that the 1'-phosphate interacts with side chains of Arg501 and Trp494 and the 6'-phosphate interacts with Thr444, Thr446, Ser449 (i.e., the 444-449 loop), and Ser531. Additionally, backbone atoms from the 527-533 loop interact with a sugar ring hydroxyl and the two effector phosphate moieties. An effector analogue series indicates that only one phosphate on the sugar is required for activation. However, singly phosphorylated sugars, including Fru-1-P and Fru-6-P, bind with a Kix in the range of 0.07-1 mM. The second phosphate of Fru-1,6-BP causes tight effector binding, because this native effector has a Kix of 0.061 μM. Glucose 1,6-bisphosphate and ribulose 1,5-bisphosphate bind in the 0.07-1 mM range. The contrast with a higher Fru-1,6-BP binding indicates specificity for the fructose sugar conformation. Site-directed random mutagenesis at each residue that contacts bound Fru-1,6-BP showed that a negative charge introduced at position 531 mimics allosteric activation, even in the absence of Fru-1,6-BP. Collectively, analogue and mutagenesis studies are consistent with the 527-533 loop playing a key role in allosteric function. Deletion mutations that shortened the 527-533 loop were expected to prevent formation of hydrogen bonds between backbone atoms on the loop and Fru-1,6-BP. Indeed, Fru-1,6-BP did not activate these loop-shortened mutant proteins. Previous structural comparisons of M1-PYK and M2-PYK indicate that the 527-533 loop makes interactions across a subunit interface when an activator is not present. Mutating the hL-PYK subunit interface interactions among Trp527, Arg528, and Asp499 mimics allosteric activation. Considered with published structures, these results are consistent with (1) the two phosphates of Fru-1,6-BP docking to Arg501/Trp494 and the 444-449 loop, respectively, and (2) the formation of hydrogen bonds among Fru-1,6-BP and backbone atoms of the 527-533 loop pulling this loop away from the subunit interface, which results in breaking of the Trp527-Arg528-Asp499 interactions to elicit an allosteric response.