Glucose-6-phosphatase specificity

Phosphatases specific for such substrates as glucose-6-phosphate, fructose-l,6-bisphosphate, and phospho-glycolate help to drive metabolic cycles (Chapter 17). The 335-residue fructose-1,6-bisphosphatase associates to form a tetramer with D2 symmetry. ° The allosteric enzyme exists in two conformational states (see Chapter 11). Activity is dependent upon Mg + or other suitable divalent cation, e.g., Mn or Zn, and is further enhanced by K+ or NH3. While the dimetal sites depicted in Figs. 12-23 and 12-24 are quite rigid and undergo little change upon formation of complexes with substrates or products, the active site of fructose-1,6-bisphosphatase is more flexible. There are three metal-binding sites but they contain no histidine side chains and have been seen clearly only in a product complex. Perhaps because of the need for... 

Two gluconeogenesis-specific phosphatases then successively cleave off the phosphate residues from fructose 1,6-bisphos-phate. In between these reactions lies the isomerization of fructose 6-phosphate to glucose 6-phosphate—another glycolytic reaction. 

The last enzyme in the pathway, g/ucose 6-phosphatase, occurs in the liver, but not in muscle. It is located in the interior of the smooth endoplasmic reticulum. Specific transporters allow glucose 6-phosphate to enter the ER and allow the glucose formed there to return to the cytoplasm. From there, it is ultimately released into the blood. 

FIGURE 20-27 Regulation of sucrose phosphate synthase by phosphorylation. A protein kinase (SPS kinase) specific for sucrose phosphate synthase (SPS) phosphorylates a Ser residue in SPS, inactivating it a specific phosphatase (SPS phosphatase) reverses this inhibition. The kinase is inhibited allosterically by glucose 6-phosphate, which also activates SPS allosterically. The phosphatase is inhibited by Pi, which also inhibits SPS directly. Thus when the concentration of glucose 6-phosphate is high as a result of active photosynthesis, SPS is activated and produces sucrose phosphate. A high P, concentration, which occurs when photosynthetic conversion of ADP to ATP is slow, inhibits sucrose phosphate synthesis. 

By analysis of the products with glucose oxidase, it was shown that the anomeric composition of the glucose liberated from glucose-6-phos-phate by the enzymes acid or alkaline phosphatase or by glucose-6-phosphatase from rat liver was essentially the same as that of the substrate, thus indicating a lack of anomeric specificity for these enzymes also (106). 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

It appears to the author that the existence of a distinct glucose-6-phosphatase is well established for endoplasmic reticulum of liver, kidney, small intestine, and pancreas but additional studies appear to be required before a similar conclusion may be reached regarding the enzyme from other sources. It should be pointed out, however, that while glucose-6-P hydrolysis by enzymes from many sources may not result from specific glucose-6-phosphatase, this hydrolysis in these tissues may nonetheless be of metabolic significance. 

The action of phosphatase on glucose-l-phosphate substrate is shown in Fig. 18. Phosphatase breaks the P-0 bond and releases the P04 group. In contrast phosphorylase which operates on the same substrate opens the C—0 bond. Apparently this affinity to specific bond sites, which in the present case amounts to a difference of ca. 1.5 A (Fig. 18) between the two bonds left and right from the oxygen, is... 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

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