Elizabeth Hubert

Univalent cation activation of fructose 1,6-diphosphatase☆

Abstract Fructose 1,6-diphosphatase from several vertebrate sources has been studied with respect to univalent cation activation. All the enzymes tested showed activation by certain univalent cations, K + or NH 4 + being the best activators. A re-evaluation of some properties of fructose 1,6-diphosphatases in the presence of univalent cation activators showed, as studied with pig kidney, rabbit liver, and rabbit muscle fructose 1,6-diphosphatase, that the Mg 2+ -saturation curves were markedly altered by the presence of 150 m m K + . Not only an increase in K a and V max was observed, but also the sigmoidal nature of the Mg 2+ -saturation curves became evident. AMP inhibition, characteristic of most fructose 1,6-diphosphatases, was not significantly altered by the presence of K + in the case of pig kidney, rabbit liver, and fish ( Raja chilensis ) liver fructose 1,6-diphosphatase. Rabbit muscle fructose 1,6-diphosphatase became more sensitive to AMP inhibition, while in the case of fish ( Genipterus chilensis ) liver fructose 1,6-diphosphatase, inhibition by AMP could only be demonstrated in the presence of the univalent cation activators.

The reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase is related to a fructose 2,6-bisphosphate allosteric site

Modification of a highly reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase with N-ethylmaleimide results in the loss of activation of the enzyme by monovalent cations. Low concentrations of fructose 2,6-bisphosphate or high (inhibitory) levels of fructose 1,6-bisphosphate protect the enzyme against the loss of monovalent cation activation, while non-inhibitory concentrations of the substrate gave partial protection. The allosteric inhibitor AMP markedly increases the reactivity of the cysteine residue. The results indicate that fructose 2,6-bisphosphate can protect the enzyme against the loss of potassium activation by binding to an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit the enzyme by binding to this allosteric site.

Suppression of Kinetic AMP Cooperativity of Fructose- 1,6-Bisphosphatase by Carbamoylation of Lysine 50

Selective treatment of pig kidney fructose 1,6-bisphosphatase with cyanate leads to the formation of an active carbamoylated derivative that shows no cooperative interaction between the AMP-binding sites, but completely retains the sensitivity to the inhibitor. By an exhaustive carbamoylation of the enzyme a derivative is formed that has a complete loss of cooperativity and a decrease of sensitivity to AMP. It was proposed that the observed changes of allosteric properties were due to the chemical modification of two lysine residues per enzyme subunit [Slebe et al. (1983), J. Protein Chem.2, 437–443]. Studies of the temperature dependence of AMP sensitivity and the interaction with Cibacron Blue Sepharose of carbamoylated fructose 1,6-bisphosphatase derivatives indicate that the lysine residue involved in AMP sensitivity is located at the allosteric AMP site, while the lysine residue involved in AMP cooperativity is at a distinct location. Using [14C]cyanate, we identified both lysine residues in the primary structure of the enzyme; Lys50 is essential for AMP cooperativity and Lys112 appears to be the reactive residue involved in the AMP sensitivity. According to the fructose 1,6-bisphosphatase crystal structure, Lys50 is strategically positioned at the C1–C2 interface, near the molecular center of the tetramer, and Lys112 is in the AMP-binding site. The results reported here, combined with the structural data of the enzyme, strongly suggest that the C1ndash;C2 interface is critical for the propagation of the allosteric signal among the AMP sites on different subunits.

Selective modification of fructose 1,6-bisphosphatase by periodate-oxidized AMP.

The gluconeogenetic enzyme, fructose 1 ,&bisphosphatase (FDPase) is composed of 4 subunits ]I]. Each subunit has an allosteric site for AMP at neutral pH [2]. The enzyme is also activated by monovalent cations such as K’ and NH: [3]. The specific inhibition of the enzyme by AMP is one of the essential regulatory mechanisms of gluconeogenesis 141. Studies on chemical modification with pyridoxal 5’-pllosphate have shown changes in the sensitivity to either allosteric AMP inhibition or to the high substrate inhibition of the enzyme [5,6]. The regulatory properties of the FDPase and the activation by monovalent cations are affected when arginine residues are modified with butanedione [7]. Here, the characteristics of the modification by adenosine .5’-monophosphate 2’3’-dialdehyde (AMP,,) as welJ as the possible invaivenlent of lysyl groups of the enzyme in the interaction with AMP and the activation by monovalent cations were examined. We show that the AMP analog can be used as an affinity probe for the allosteric site.

Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase☆

The specific chemical modification by sodium cyanate of highly reactive cysteine residues at pH 7.5 in pig kidney fructose 1,6-bisphosphatase results in the reversible loss of activation of the enzyme by monovalent cations. No loss of activation by potassium ions occurs when modification is carried out in the presence of fructose 2,6-bisphosphate. The effect of Mg2+ on native and cyanate-modified enzyme activities implicates the above cysteine residue as being directly linked to the inhibition by both the divalent cation and fructose 2,6-bisphosphate. Incorporation of [14C]cyanate to the enzyme shows that the blockage of two reactive residues per tetramer is sufficient to eliminate the activation of the enzyme by K+.

Fructose 1,6-bisphosphatase: Dissociation of AMP cooperativity and AMP inhibition by carbamylation

Selective treatment of pig kidney fructose 1,6-bisphosphatase with potassium cyanate leads to the formation of an active carbamylated enzyme that has lost the cooperative interactions among AMP sites, but retains sensitivity to inhibition of catalytic activity by the regulator AMP. Incorporation data on [14C]KNCO indicate that the loss of enzyme cooperativity at the AMP sites is related to selective carbamylation of four lysine residues per mole of tetrameric enzyme. Exhaustive carbamylation suggests that a second lysine residue per subunit is essential for AMP inhibition.

Naphthyl phosphates as substrates for fructose 1,6-diphosphatase.

Mammalian liver and kidney fructose l,Bdiphosphatase (EC 3.1.3.11) (FDPase) is considered to be a highly specific enzyme for the hydrolysis of the l-phosphate from fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate [ 11. However, under appropriate conditions FDPase also hydroloyzes at reduced rates several other phosphate esters: phosphoenol pyruvate [ 21, fi-glycerol phosphate [ 31, p-nitrophenyl phosphate [4], and fructose-l-phosphate [ 51. To a certain extent, the enzyme could be regarded as a nonspecific phosphatase with highly increased affinities and catalytic activities for two substrates: fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate. This view led us to study the reaction of FDPase with naphthyl phosphates, since the hydrolysis of these well established substrates for most non-specific phosphatases [6-81 could be used for detection of FDPase on polyacrylamide gels.