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A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity.

ABSTRACT: A variant form of yeast pyruvate kinase (EC 2.7.1.40) with Ser-384 mutated to proline has been engineered in order to study the allosteric properties of this enzyme. Both the mutant and wild-type enzymes were overexpressed in a strain of yeast in which the genomic copy of the pyruvate kinase gene had been disrupted by an insertion of the Ura3 gene. Both enzymes were purified to homogeneity and their kinetic properties characterized. The wild-type enzyme displays sigmoid kinetics with respect to phosphoenolpyruvate (PEP) concentration, and is activated by the allosteric effect fructose 1,6-bisphosphate with concomitant reduction in co-operativity. In contrast, the mutant was found to be dependent on the presence of the effector for catalytic activity and was inactive in its absence. The fully activated mutant enzyme had a kcat. 1.6 times greater than that of the wild-type enzyme. The mutation introduced into the enzyme is in an intersubunit contact which is known to be critical for the allosteric properties of the enzyme, and is far removed from the active site. The major effect of the mutation seems to be to stabilize the low-affinity T state of the apoenzyme, although kcat. is also affected. The S0.5 for PEP and S0.5 for ADP of the wild-type enzyme were 0.22 +/- 0.004 and 0.15 +/- 0.01 mM respectively (means +/- S.E.M.). In the activated mutant enzyme, these kinetic parameters increased to 0.67 +/- 0.03 and 0.43 +/- 0.03 mM respectively. The cooperativity between ADP-binding sites was altered in the mutant enzyme, with the Hill coefficient (h) for ADP increasing to 1.65 +/- 0.07 in the presence of the effector, compared with a value of 0.01 +/- 0.07 for the wild-type enzyme under the same conditions. CD spectroscopy revealed the secondary structure of the mutant enzyme to be little different from that of the wild-type enzyme, indicating that the two enzymes have similar secondary structures in solution. Precise tertiary and quaternary structures such as intersubunit and interdomain interactions may be modified. An improved purification procedure has been devised that allows large quantities of enzyme to be rapidly prepared.

SUBMITTER: Collins RA

PROVIDER: S-EPMC1135862 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description: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 ?M. 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.

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A subunit interface mutant of yeast pyruvate kinase requires the allosteric activator fructose 1,6-bisphosphate for activity.

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