Allosteric interactions in phosphorylase B

An allosteric model of phosphorylase B is proposed based on the following assumptions. The enzyme consists of two sub-units and undergoes a concerted transition from the inactive T to the active R state. The binding of substrates, phosphate, and glycogen is regarded as exclusive, but the binding of the activator AMP is nonexclusive. The enzyme model is of the K, V type, i. e., the activator AMP is important, not only for the T-R transition and the substrates binding, but also for the formation of the active site. Therefore, it displays a big influence on the maximal reaction rate. Calculations based on this model lead to an equation containing 5 constants, which can be easily computed from kinetic data. All kinetic measurements fit the expressions derived from the model. Independent methods for the measurement of all the constants involved were developed. They are based on the study of binding of phosphorylase with the substrates and the activator. These measurements are in satisfactory agreement with the data obtained from enzyme kinetics.     

Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b.

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is a trimeric enzyme that readily dissociates to the monomer. Dilution of enzyme from 20 to 0.02 microgram of protein/ml is accompanied by a greater than 50-fold increase in the specific activity (vtrimer = 0.23 nmol/min/microgram; vmonomer = 12.5 nmol/min/micrograms). Gel permeation chromatography in the presence of the substrate phosphate shows the enzyme to be predominantly trimeric at 50 mM Pi and predominantly monomeric at 100 mM Pi, when experiments are done at 24 degrees C. No significant dissociation was observed at 4 degrees C with Pi or at either temperature with the substrate inosine. As measured by dissociation, the L0.5 for Pi is 88 mM and thus significantly higher than the Km of 3.1 mM for Pi. Enzyme activity as a function of phosphate concentration showed negative cooperativity, but the conformational response measured by the change in native Mr during dissociation showed positive cooperatively toward Pi. These data support a model for two separate phosphate binding sites on the enzyme. The activity and stability of purine nucleoside phosphorylase are quite sensitive to the concentration of the enzyme as well as appropriate substrates. Although the monomer is interpreted as being a fully active form of the enzyme, the data in general are most consistent with the enzyme functioning in vivo as a regulated trimer.     

Effect of sodium cholate on the catalytic and structural properties of phosphorylase b

Sodium cholate at millimolar concentration is able to induce activity in rabbit muscle phosphorylase b in the absence of AMP. The maximum activation of the enzyme in presence of 7 mM sodium cholate was 24% of that achieved by 1 mM AMP. Other bile salts tested showed a negligible activating effect. The Ka for AMP was lowered fivefold by 5 mM of the steroid detergent, while the cooperative binding of the nucleotide was abolished. Phosphorylase b', a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack, presented an activation profile similar to that of phosphorylase b. In contrast, phosphorylase a was inhibited by the bile salt, while the activity of liver phosphorylase b was not significantly affected. Modification of the AMP site of the enzyme with 2,3-butanedione could not inhibit sodium-cholate-induced activity. tert-Butanol, an organic solvent activator of phosphorylase b, was found to enhance the activity induced by sodium cholate. The interaction of sodium cholate and phosphorylase b was also followed by difference spectroscopy using a fluorescein isothiocyanate--phosphorylase b conjugate. Furthermore, measurements of electron spin resonance demonstrated that the mobility of a spin-label bound at buried--NH2 groups of phosphorylase b decreases cooperatively with increasing bile salt concentration.     

Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy

The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.